Lorenzo Pastrana Castro

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.

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.

Antimicrobial activities of the bacteriocin-like substances produced by lactic acid bacteria isolated from Moroccan dromedary milk

A total of 450 different colonies, isolated from 25 samples of dromedary milk collected from Laâyoune region of Morocco, were tested for antimicrobial compounds production. Out of these, 30 were determined to be lactic acid bacteria (LAB) and able to inhibit the growth of the indicator strain Listeria innocua CECT 4030. Seven isolates were selected by the large and clear zones of inhibition when tested by the agar well diffusion assay. They were classified by phenotypic and biochemical analysis as two Enterococcus durans (E204 and E214), two Lactococcus lactis (R75 and R76), one Enterococcus faecium R111, one Lactococcus cremoris R112 and one Enterococcus avium R122. Their antimicrobial compounds were detected in cell-free culture supernatant fluids under conditions that eliminate acid and hydrogen peroxide inhibition. The antimicrobial activity was altered after treatment with trypsin, -chymotrypsin, pepsin or papain which confirms the proteinaceous nature of the inhibition. It was heat stable even at autoclaving temperature (121°C for 15 min) and also active over a wide pH range (2 to 10). This fact suggests that bacteriocin-like produced by the seven LAB strains may find application as biopreservatives in food products. Key words: Dromedary milk, lactic acid bacteria, bacteriocin-like substances, antimicrobial activity.

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.

Production and Characterization of Distilled Alcoholic Beverages Obtained by Solid-State Fermentation of Black Mulberry (Morus nigra L.) and Black Currant (Ribes nigrum L.)

The present study was conducted to appraise the potential of black mulberry and black currant to be used as fermentation substrates for producing alcoholic beverages obtained by distillation of the fruits previously fermented with Sacchromyces cerevisiae IFI83. In the two distillates obtained, the volatile compounds that can pose health hazards are within the limits of acceptability fixed by the European Council (Regulation 110/2008) for fruit spirits. However, the amount of volatile substances in the black currant distillate (121.1 g/hL absolute alcohol (aa)) was lower than the minimum limit (200 g/hL aa) fixed by the aforementioned regulation. The mean volatile composition of both distillates was different from other alcoholic beverages such as four commercial Galician orujo spirits, Portuguese bagaceiras, and two distillates obtained from fermented whey and blackberry. The results obtained showed the feasibility for obtaining distillates from fermented black mulberry and black currant, which have their own distinctive characteristics.

Performance and intestinal coliform counts in weaned piglets fed a probiotic culture (Lactobacillus casei subsp. casei CECT 4043) or an antibiotic.

The production of biomass and antibacterial extracellular products by Lactobacillus casei subsp. casei CECT 4043 was followed in both batch and in realkalized fed-batch cultures. Enhanced concentrations of biomass and antibacterial extracellular products were obtained with the use of the latter fermentation technique in comparison with the batch mode. The culture obtained by fed-batch fermentation was mixed with skim milk and used to prepare a probiotic feed for weaned piglets. To test the effect of the potentially probiotic culture of L. casei on body weight gain, feed intake, feed conversion efficiency, and on fecal coliform counts of piglets, two groups of animals received either feed supplemented with the probiotic preparation or avilamycin for 28 days. The control group was fed nonsupplemented feed. At the end of the administration period (day 28), the groups receiving probiotic and avilamycin exhibited the highest average body weight gain values, although the mean feed intake and feed conversion efficiency values were not different among the groups (P > 0.05). For the entire experimental period (42 days), the control group exhibited the lowest feed intake value, the probiotic group exhibited the highest feed conversion efficiency value, and the antibiotic group exhibited the highest body weight gain (P 0.05). Fecal coliform values decreased (although not significantly) by day 28 in the three groups. However, the mean counts returned to pretreatment levels by day 42 in all groups.

Growth of Enterococcus durans E204 producing bacteriocin-like substance in MRS Broth: Description of the growth and quantification of the bacteriocin-like substance

Bacteriocin-like substance E204 is an antimicrobial compound produced by Enterococcus durans E204 isolated from camel milk of Morocco that shows a broad spectrum of inhibitory activity against taxonomically related microorganisms. It is sensitive to digestive proteases. In the first study, de Man, Regosa and Sharpe (MRS) broth was inoculated by E204 strain, incubated for 30°C at 200 rpm and monitored by checking dry cell weight, nutrients consumption, lactic acid and bacteriocin-like productions. The maximum biomass (2.3 g/l) and antimicrobial activity (32 AUml -1 ) were obtained at 12 h of incubation. No increase in the production of bacteriocin-like was recorded after the exponential phase. In the other hand, the quantification of the antimicrobial activity was carried out by a photometric assay on culture tubes based on the determination of the ID50 dose causing 50% growth inhibition of Enterococcus faecium 410 CECT in 6 h of incubation. The highest bacteriocin-like titre (279.71 BUml -1 ) was obtained at acidic pH (3.5) and at 70°C after 10 min of incubation. Key words : Enterococcus durans, bacteriocin-like, MRS broth, nutrients consumption, quantification.

Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P < 0.05) and K M increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G].