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Three bifidobacteria strains of human origin (Bifidobacterium pseudolongum INIA P2, Bifidobacterium breve INIA P734, and Bifidobacterium longum INIA P678) were used as potential probiotic adjunct cultures for the manufacture of pasteurized sheep milk cheese. Bifidobacteria were inoculated at 5 to 6 log cfu/mL in milk vats. Microbiological, physicochemical, rheological, color, and sensory characteristics were determined at 7, 28, and 60 d of ripening. Counts of B. pseudolongum INIA P2 remained above 6 log cfu/g during 60 d of ripening as well as after further simulated gastrointestinal digestion of cheeses. Bifidobacterium breve INIA P734 counts remained stable during 28 d and decreased by less than 1 log unit after simulated digestion. Bifidobacterium longum INIA P678 counts dropped sharply during cheese manufacture and ripening and were below detection level after simulated digestion. Addition of bifidobacteria strains did not affect starter viability, cheese pH, dry matter, water activity, or salt content but significantly increased overall proteolysis and the concentration of some free amino acids. Cheeses with bifidobacteria exhibited no significant differences in most sensory characteristics with respect to control cheese. According to our results, B. breve INIA P734 and B. pseudolongum INIA P2 are promising candidates as probiotic adjunct cultures in fresh and semi-hard sheep milk cheese.

The publisher regrets the incorrect publication of Tables in the original article. In the title of Tables 3, 4, 5, 6 and 7 “L. lactis subsp. lactis INIA 415” should be “L. lactis subsp. lactis INIA 415” and “C. tyrobutyricum CECT 4011” should be “C. tyrobutyricum CECT 4011”. In the Tables 4, 5, 6 and 7 the headings of the columns are incorrect, the line below heading ‘Spores of C. tyrobutyricum CECT 4011’ should be partial, running from the 2nd to the 8th results columns (vats 2–8). Therefore, the first results column, 1% INIA 415–2 (vat 1, the negative control of late blowing defect), did not receive any spores. The tables are now displayed correctly below. The publisher would like to apologise for any inconvenience caused.

Lipid oxidation and proteolysis are essential processes in Serrano dry-cured ham quality. The influence of high pressure processing (HPP) at 600 MPa for 6 min on lipid oxidation, aminopeptidase (AP) activities and free amino acids (FAA) in ripened Serrano hams of different chemical composition after 5 months at 4 °C were studied. HPP increased lipid peroxidation indexes. Composition influenced both indexes, with higher levels in hams of medium or high intramuscular fat (IMF) content and in hams of low or medium salt content or salt-in-lean ratio. HPP lowered AP activities by more than 50%. Composition also affected AP activities, with lower levels in hams of low aw, high IMF content, low salt content or low salt-in-lean ratio. At the end of refrigerated storage, HPP only affected Arg and Tyr levels. Many of the individual FAA reached higher levels in hams of low aw, medium or high IMF content, low or medium salt content, or low or medium salt-in-lean ratio.

Three strains with technological and probiotic properties (Lactococcus lactis INIA 650, Lacticaseibacillus paracasei INIA P272 and Bifidobacterium breve INIA P734) were used in different combinations as adjunct cultures for the manufacture of pasteurized ewe's milk. The three strains showed good viability during cheese manufacture and ripening. Moreover, they all were able to survive after the addition of the cheese samples to an in vitro colonic model. The nisin-producing strain L. lactis INIA 650 was able to produce the antimicrobial peptide nisin not only during cheese manufacture and ripening, but also in the colonic model. None of the strains combinations added to the cheese as adjunct cultures had a negative influence in their physicochemical or sensory characteristics when compared to control cheese. This work shows the suitability of cheese as vehicle for a multi-strain combination with potential dual effect in food and gut.

Isoflavones intake is associated with health benefits. The metabolism of isoflavones by bacteria plays a key role in their biotransformation. Therefore, commercial soy drink was fermented by 11 lactic acid bacteria (LAB) and 9 bifidobacteria strains. The majority of the strains showed deglycosylation of the isoflavone glycosides present in soy drink and appearance of the aglycones daidzein, genistein and glycitein. Moreover, we observed the further transformation of daidzein into O-desmethylangolensin (O-DMA) and tetrahydrodaidzein, alongside with dihydrodaidzein (DHD) and a putative isomer of DHD. On the other hand, genistein was transformed by nearly all strains into 6-hydroxy-O-desmethylangolensin (6-hydroxy-O-DMA), but no dihydrogenistein production was registered. A high concentration of 2-(4-hydroxyphenyl)-propionic acid was observed, suggesting the degradation of O-DMA and 6-hydroxy-O-DMA. The potential of LAB and Bifidobacterium strains to produce functional soy drink enriched with bioactive isoflavones is demonstrated in this work.

The effect on the biochemical characteristics of Ibérico semi-hard cheese supplemented with Himanthalia elongata (HE), Laminaria ochroleuca (LO), Porphyra umbilicalis (PU), Ulva lactuca (UL) or Undaria pinnatifida (UP) seaweeds was investigated. Addition of 1% dehydrated seaweed to curd increased total free amino acids in LO cheese and decreased them in HE and UP cheeses at 60 days with respect to control cheese. Lipolysis was markedly enhanced in UL cheese, which showed on day 60 a 6.0-fold higher concentration of total free fatty acids than control cheese. Seventy-six volatile compounds were detected in cheeses with seaweeds and 54 in control cheese. Aldehydes and alcohols reached higher levels in the control cheese, esters and ketones in PU cheese, acids and sulphur compounds in UL cheese, hydrocarbons in HE and PU cheeses, and furans in HE and UP cheeses.

Healthy effect of isoflavones consumption is attributed to their bioactive metabolites such as O-desmethylangolensin (O-DMA) produced by intestinal bacteria. Evidence from in vitro studies suggests that O-DMA has several cancer-related biological actions. Enterococcus spp. are indigenous species of the human gastrointestinal tract. Significant attention has been focused on Enterococcus faecium strains with probiotic and /or technological interest in foods. In this work, we describe the production of the bioactive isoflavones O-DMA and 6-hydroxy O-DMA by the strain E. faecium INIA P553. O-DMA was produced from dihydrodaidzein, daidzein, daidzin and formononetin, and 6-hydroxy O-DMA from dihydrogenistein, genistein and genistin with high efficiency. To our knowledge, it is the first bacterium with probiotic potential capable of producing O-DMA and 6-hydroxy O-DMA. Therefore, this strain shows potential applications in biotechnology in functional foods and as probiotic.

Urolithins are microbial metabolites derived from ellagic acid by the action of intestinal bacterial. Potential beneficial effects of urolithins A and B on certain types of cancer and on muscular health during aging have been demonstrated. In this work, urolithin production by 48 strains of bifidobacteria and 1070 bacterial isolates from the faeces of a healthy woman whose intestinal microbiota produced uroltihins A and B was screened. Bifidobacterium pseudocatenulatum INIA P815 was the only strain able to produce urolithins A and B from ellagic acid. Influence of different media on the strain metabolism showed that the production of urolithins could be regulated. The identification of a bifidobacterial strain producing urolithins A and B opens the possibility for its application as probiotic in functional foods and in the development of nutraceuticals.

Healthy effect of isoflavones consumption is attributed to their bioactive metabolites such as O-desmethylangolensin (O-DMA) produced by intestinal bacteria. Evidence from in vitro studies suggests that O-DMA has several cancer-related biological actions. Enterococcus spp. are indigenous species of the human gastrointestinal tract. Significant attention has been focused on Enterococcus faecium strains with probiotic and /or technological interest in foods. In this work, we describe the production of the bioactive isoflavones O-DMA and 6-hydroxy O-DMA by the strain E. faecium INIA P553. O-DMA was produced from dihydrodaidzein, daidzein, daidzin and formononetin, and 6-hydroxy O-DMA from dihydrogenistein, genistein and genistin with high efficiency. To our knowledge, it is the first bacterium with probiotic potential capable of producing O-DMA and 6-hydroxy O-DMA. Therefore, this strain shows potential applications in biotechnology in functional foods and as probiotic.

Urolithins are microbial metabolites derived from ellagic acid by the action of intestinal bacterial. Potential beneficial effects of urolithins A and B on certain types of cancer and on muscular health during aging have been demonstrated. In this work, urolithin production by 48 strains of bifidobacteria and 1070 bacterial isolates from the faeces of a healthy woman whose intestinal microbiota produced uroltihins A and B was screened. Bifidobacterium pseudocatenulatum INIA P815 was the only strain able to produce urolithins A and B from ellagic acid. Influence of different media on the strain metabolism showed that the production of urolithins could be regulated. The identification of a bifidobacterial strain producing urolithins A and B opens the possibility for its application as probiotic in functional foods and in the development of nutraceuticals.

Clostridium spore counts, sugars and organic acids were investigated in 52 Manchego cheeses with different degrees of late blowing defect (LBD) and in 25 Manchego cheeses without LBD. Cheeses with LBD showed significantly higher spore counts, lower levels of lactose, galactose, citric and lactic acids, and higher levels of acetic and butyric acids than cheeses without LBD. The same effect of LBD on sugars and organic acids, with the exceptions of galactose and propionic acid, was recorded for raw milk (RM) and pasteurized milk (PM) Manchego cheeses. Galactose concentration was significantly lower in PM cheeses with LBD than in PM cheeses without LBD, but no significant differences were found between RM cheeses with and without LBD. Propionic acid content was significantly lower in RM cheeses with LBD, and significantly higher in PM cheeses with LBD, than in the respective cheeses without LBD. © 2012 Elsevier Ltd.

Isoflavones are usually found in nature in their glycosilated or methylated forms, and should be hydrolysed to become bioavailable and physiologically active. The deglycosylation of isoflavone C-glycosides and O-glycosides and the demethylase activity were studied in a selection of lactic acid bacteria (LAB) and bifidobacteria by assessing the degree of transformation of the pure precursor compounds, daidzin, genistin, puerarin, formononetin and biochanin A into daidzein or genistein. Only one Bifidobacterium strain and two Enterococcus strains hydrolysed the C-glycosidic bond of puerarin, while deglycosylation of O-glycosides daidzin and genistin was observed in all the tested strains. Demethylation of biochanin A and formononetin was observed in the most of LAB and bifidobacteria. Besides, the subsequent metabolites dihydrodaidzein and dihydrogenistein where produced by many of the strains via daidzein and genistein. In this work, we show the potential of LAB and bifidobacteria as part of functional foods because of their ability to transform plant isoflavones into their bioactive forms. © 2017 Elsevier Ltd

The suitability of the biopreservation system formed by reuterin-producing L. reuteri INIA P572 and glycerol (required for reuterin production) to prevent late blowing defect (LBD) was evaluated in in_x0002_dustrial sized semi-hard ewe milk cheese contaminated with Clostridium tyrobutyricum INIA 68, a wild strain isolated from a LBD cheese. For this purpose, six batches of cheese were made (three with and three without clostridial spores): control cheeses with lactococci starter, cheeses with L. reuteri as adjunct, and cheeses with L. reuteri and 30 mM glycerol. Spores of C. tyrobutyricum INIA 68 germinated during pressing of cheese curd, causing butyric acid fermentation in cheese after 30 d of ripening. The addition of L. reuteri, without glycerol, enhanced the symptoms and the formation of volatile compounds associated with LBD. When glycerol was added to cheese milk contaminated with C. tyrobutyricum, L. reuteri was able to produce reuterin in cheese resulting in cheeses with a uniform cheese matrix and a volatile profile similar to cheese made with L. reuteri and glycerol (without spores). Accordingly, L. reuteri INIA P572 coupled with glycerol seems a novel biopreservation system to inhibit Clostridium growth and prevent LBD by means of in situ reuterin production.

High pressure (HP) offers potential industrial applications in cheese preservation, but it is essential to provide knowledge concerning their effects on the ripening process and sensory characteristics. In this study, we in_x0002_vestigated the effect of different HP treatments (200–500 MPa at 14 °C for 10 min on day 7) on proteolysis, texture, colour, volatile compounds and sensory characteristics of semi-hard raw ewe milk cheese. HP treatments did not affect pH or dry matter values of 60-day-old cheeses. Treatments at pressure levels up to 400 MPa led to significant (P < 0.01) increases in the total free amino acids (FAA) content at 60 days, compared to control cheese, although the cell-free aminopeptidase activity was lower. HP retarded the formation of some volatile compounds in cheeses, the number of compounds affected by HP being higher as the pressure level increased. Cheeses pressurized at 300–500 MPa had lower levels of 2-butanone, 2-butanol, 2-propen-1-ol, 1-butanol and acetic acid than control cheese, cheeses pressurized at 400–500 MPa lower levels of 1-propanol, 2-pentanol, and butyric and hexanoic acids, and cheeses pressurized at 500 MPa lower levels of ethanol, 3-methyl-1-butanol and 3-methyl-2-buten-1-ol. All HP-treated cheeses showed higher fracturability values, and higher Hue angle and lower a* values than control cheese. Despite the differences detected by instrumental analyses between HP_x0002_cheeses and control cheese, few significant differences were found between the sensory characteristics of HP_x0002_cheeses and control cheese. Only the pressurization of cheese at 500 MPa affected significantly (P < 0.01) some of the sensory characteristics, with a negative effect on taste intensity but a positive effect on aroma quality. In summary, HP treatments at 200 and 300 MPa showed the mildest effects on the characteristics of semi-hard raw ewe milk cheese. HP treatment of this cheese variety at 300, 400 and 500 MPa prevented late blowing defect ca

In this study we evaluated the application of different high pressure (HP) treatments (200–500 MPa at 14 °C for 10 min) to industrial sized semi-hard cheeses on day 7, with the aim of controlling two Clostridium tyrobutyricum strains causing butyric acid fermentation and cheese late blowing defect (LBD). Clostridium metabolism and LBD appearance in cheeses were monitored by sensory (cheese swelling, cracks/splits, off-odours) and instrumental analyses (organic acids by HPLC and volatile compounds by SPME/GC–MS) after 60 days. Cheeses with clostridial spores HP-untreated and HP-treated at 200 MPa showed visible LBD symptoms, lower concentrations of lactic, citric and acetic acids, and higher levels of pyruvic, propionic and butyric acids and of 1-butanol, ethyl and methyl butanoate, and ethyl pentanoate than cheeses without spores. However, cheeses with clostridial spores and HP-treated at ≥ 300 MPa did not show LBD symptoms and their organic acids and volatile compounds profiles were comparable to those of their respective HP-treated control cheeses, despite HP treatments caused a low spore reduction. A decrease in C. tyrobutyricum spore counts was observed after curd pressing, which seems to indicate an early spore germination, suggesting that HP treatments ≥300 MPa were able to inactivate the emerged C. tyrobutyricum vegetative cells and, thereby, prevent LBD. © 2016 Elsevier Ltd

The biochemical, physical and sensory characteristics of ewe milk cheeses made with reuterin-producing Lactobacillus reuteri and glycerol (substrate for reuterin production) were assessed. Cheese made with lactococci starter (CTRL), cheese made with starter and L. reuteri (SLR), and cheese made with starter, L. reuteri and 30�mM glycerol (SLR-G) were manufactured. L. reuteri reached counts above 7�log�cfu/g on day 1. Lactococci survival was enhanced in SLR cheese without affecting cheese pH, dry matter, proteolysis, concentration of most free amino acids (FAA), textural and most color parameters, or sensory characteristics. In situ production of reuterin by L. reuteri was only detected in SLR-G cheese, decreasing LAB counts although acidification remained unaffected. SLR-G cheese showed higher values of cell free aminopeptidase activity, overall proteolysis and FAA, particularly glutamic acid, than CTRL and SLR cheeses. The addition of L. reuteri-glycerol resulted in lower hardness and elasticity values in SLR-G cheese and influenced its L*, a* and b* color parameters. However, these changes, which were detected by instrumental analysis, did not affect the sensory scores for texture and color quality of SLR-G cheese, and it received the highest scores for taste quality. Our results suggest that L. reuteri-glycerol may provide a suitable system to release the antimicrobial reuterin in cheese without affecting negatively its sensory characteristics. � 2016 Elsevier Ltd

Reuterin has a high potential as a food preservative due to both its chemical characteristics and its antimicrobial activity against food-borne pathogens and spoilage bacteria. However, there is a lack of information about its toxicity and its capacity to interfere with the metabolism of drugs by inhibiting cytochrome P450 (CYP) activity. The results of this study indicated that reuterin exhibited a moderate cytotoxicity in the human hepatoma cell line HepG2 according to assays measuring three different endpoints in the same set of cells. Reuterin was much less toxic than acrolein and only four times more toxic than diacetyl, a generally recognized as safe flavoring compound. In vitro experiments utilizing human liver microsomes showed that reuterin presents low possibility of displaying in vivo drug interactions by inhibition of CYP3A4, CYP2D6, and CYP2C9. Therefore, reuterin can be considered a promising food biopreservative, although additional toxicology research is needed before permission for use can be granted. © 2016 Elsevier Ltd

Brie cheeses were high pressure (HP)-treated at 400 or 600MPa on days 14 or 21 after manufacture to prevent over-ripening. Lactic acid bacteria and Penicillium camemberti numbers declined markedly after HP treatment. In control cheese pH increased 2.0 units from day 21 to day 60, but less than 0.3 units in HP-treated cheeses. Cheeses treated at 600MPa showed the maximum concentrations of residual caseins during refrigerated storage and control cheese the minimum concentrations. A 7.6-fold increase in hydrophobic peptides was recorded from day 21 to day 60 in control cheese and 0.8-1.6-fold increases in HP-treated cheeses. The maximum aminopeptidase activity was detected in control cheese, the highest free amino acid concentrations in cheeses treated at 400MPa. The firmest texture was recorded for cheeses treated on day 14 at 400 or 600MPa. HP-treated cheeses showed higher flavour quality scores than control cheese from day 60 onwards. © 2014 Elsevier Ltd.

In this study, reuterin-producing Lactobacillus reuteri INIA P572 was added to cheese as an adjunct culture together with 50 or 100mM glycerol (required for reuterin production), with the aim of controlling Clostridium tyrobutyricum CECT 4011 growth and preventing the late blowing defect (LBD) of cheese caused by this strain. L.reuteri survived cheese manufacture and produced reuterin in situ, detected at 6 and 24h. However, the produced reuterin was enough to inhibit the growth of Clostridium, showing undetectable spore counts from day 30 onward and, therefore, to prevent cheese LBD during ripening (60d, 14°C). The acidification of these cheeses was not affected, although from day 14 they showed significantly lower lactococci counts than cheese made only with the starter (control cheese). Cheeses with LBD showed lower levels of lactic acid than control cheese and the formation of propionic and butyric acids, but cheeses with reuterin showed the same organic acids profile than control cheese. The cheese made with L.reuteri and 100mM glycerol showed a light pink colour, not observed in the cheese made with L.reuteri and 50mM glycerol. These results demonstrated a potent anti-clostridial activity of reuterin produced in an actual food product like cheese, and proved to be a novel approach to prevent LBD of cheese. © 2014 Elsevier Ltd.

Enzyme-rich cheeses are prone to over-ripening during refrigerated storage. Blue-veined cheeses fall within this category because of the profuse growth of Penicillium roqueforti in their interior, which results in the production of highly active proteinases, lipases, and other enzymes responsible for the formation of a great number of flavor compounds. To control the excessive formation of free fatty acids (FFA) and volatile compounds, blue-veined cheeses were submitted to high-pressure processing (HPP) at 400 or 600. MPa on d 21, 42, or 63 after manufacture. Cheeses were ripened for 30. d at 10°C and 93% relative humidity, followed by 60. d at 5°C, and then held at 3°C until d 360. High-pressure processing influenced the concentrations of acetic acid and short-chain, medium-chain, and long-chain FFA. The effect was dependent on treatment conditions (pressure level and cheese age at the time of treatment). The lowest concentrations of acetic acid and FFA were recorded for cheeses treated at 600. MPa on d 21; these cheeses showed the lowest esterase activity values. Acetic acid and all FFA groups increased during ripening and refrigerated storage. The 102 volatile compounds detected in cheese belonged to 10 chemical groups (5 aldehydes, 12 ketones, 17 alcohols, 12 acids, 35 esters, 9 hydrocarbons, 5 aromatic compounds, 3 nitrogen compounds, 3 terpenes, and 1 sulfur compound). High-pressure processing influenced the levels of 97 individual compounds, whereas 68 individual compounds varied during refrigerated storage. Total concentrations of all groups of volatile compounds were influenced by HPP, but only ketones, acids, esters, and sulfur compounds varied during refrigerated storage. The lowest total concentrations for most groups of volatile compounds were recorded for the cheese pressurized at 600. MPa on d 21. A principal component analysis combining total concentrations of groups of FFA and volatile compounds discriminated cheeses by age and by the pressure level

The effect of high hydrostatic pressure (HHP) processing at 250 and 450 MPa for 10 min combined with the lactoperoxidase system (LPS) on the inactivation of Listeria monocytogenes H66a and the characteristics of cold-smoked salmon during 35 d at 5°C were investigated. A synergistic antimicrobial effect of 450 MPa and LPS against L. monocytogenes was registered, preventing the pathogen recovery. Biogenic amine formation was avoided. Lightness (L*) values increased by HHP and LPS treatments applied individually or in combination, resulting in a brighter and less transparent appearance of smoked salmon. Redness (a*) and yellowness (b*) were also affected. Hardness and shear strength increased with HHP processing and LPS treatments. HHP at 450 MPa for 10 min in combination with the LPS added to smoked salmon might be used as a hurdle technology approach against L. monocytogenes, increasing the safety and the shelf-life during refrigerated storage. Industrial relevance Antimicrobial activity against L. monocytogenes of high pressure treatments in combination with a biopreservative, the lactoperoxidase system in refrigerated cold-smoked salmon is presented. Color and texture of cold-smoked salmon were affected, whereas the safety and shelf-life of the product were improved. © 2012 Elsevier Ltd.

Late blowing defect (LBD) is caused by the outgrowth in cheese of strains of the genus Clostridium capable of fermenting lactic acid with production of butyric acid, acetic acid, carbon dioxide and hydrogen, resulting in the appearance of texture and flavour defects. In the present work, 223 Clostridium isolates (C. beijerinckii, C. butyricum, C. sporogenes and C. tyrobutyricum) from 45 Manchego cheeses with LBD were typed by a modified short pulsed field gel electrophoresis (PFGE) protocol that solved several problems of the original protocol such as DNA degradation, incomplete DNA digestion and reduced DNA release. Our modified protocol allowed the typification of all isolates and the study of their genetic diversity. Out of the 84 Clostridium pulsotypes found in Manchego cheeses with LBD, 54 pulsotypes were represented by more than one isolate. Isolates of the same pulsotype were found in the same cheese for 36 of those 54 pulsotypes, in different cheeses from the same factory for 5 pulsotypes, and in cheeses from different factories for 13 pulsotypes. Dates of manufacture of the cheeses from the same factory containing the same pulsotype differed by less than 40 days. Thirty-six out of the 45 cheeses with LBD contained more than one Clostridium pulsotype. Clostridium isolates from Manchego cheeses with LBD showed a high genetic diversity and could be grouped by their pulsotype according to the factory and the date of cheese manufacture. © 2012 Elsevier Ltd.

The effect of high pressure (HP) on Salmonella Enteritidis in sliced dry-cured ham stored under temperature abuse (8 °C) during 60. d was investigated. After treatment, reductions of S. Enteritidis were 1.06, 2.54 and 4.32 log units in ham treated at 400, 500 and 600. MPa for 5. min at 12 °C, compared to non-pressurized samples. After 60. d, counts of S. Enteritidis in ham treated at 400 and 500. MPa were 2.56 and 2.66 log units lower than in non-treated ham, whereas the pathogen was only detected after enrichment in ham treated at 600. MPa. Lipid oxidation increased with storage and pressurization, whereas total free amino acid contents were similar in HP and control samples after 60. d. Dry-cured ham treated at the highest pressures exhibited lower shear resistance, whereas the maximum force to compress the sample was slightly changed. Color (L*, a* and b*) varied with pressurization and storage. Changes induced by HP in dry-cured ham were attenuated during storage. © 2012 Elsevier Ltd.

Hispánico cheese is a semihard variety made from a mixture of cow and ewe milks. Production of ewe milk declines in summer and autumn. To surmount the seasonal shortage of ewe milk and prevent the inactivation of milk enzymes by pasteurization, curd made in spring from ewe raw milk was pressurized at 200 and 300. MPa and stored frozen for 4 mo. Thawed ewe milk curds were added to fresh curd made from pasteurized cow milk for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same proportions as those used for experimental cheeses. Experimental cheeses exhibited lower dry matter content, higher aminopeptidase activity and total free amino acid concentration, and higher levels of acetic and propionic acids, aldehydes, alcohols, and esters compared with control cheese. In contrast, the concentration of total free fatty acids and ketones and the levels of textural parameters were significantly higher in control cheese. The use of ewe raw milk curd pressurized at 200 and 300. MPa, stored frozen and thawed for Hispánico cheese manufacture, was generally beneficial for cheese characteristics and increased cheese yield because of the lower dry matter content of experimental cheeses. © 2012 American Dairy Science Association.

The effect of high pressure (HP) treatments on microbiological, chemical, textural and sensory characteristics of vacuum-packaged cold-smoked cod refrigerated at 5 °C for 60 d was evaluated. HP at 400, 500 and 600 MPa for 5 and 10 min reduced microbial counts of smoked cod and delayed microbial growth during refrigerated storage. No differences in lipid oxidation estimated as thiobarbituric acid reactive substances (TBARS) were observed after pressurization and during refrigeration. Only tryptamine and spermine at very low levels were detected among biogenic amines in smoked cod. High pressure treatments induced a brighter appearance of smoked cod, with higher values of L* and b*, whereas a* values decreased with pressurization. HP affected hardness and shear strength of smoked cod, with higher values after treatment, but differences with untreated smoked cod were attenuated during the storage period. No differences in overall appearance and odor of smoked cod were reported after sensory evaluation. The results obtained in the present work point to the usefulness of pressurization of smoked cod at 400 MPa for 10 min or 500 MPa for 5 min to obtain an acceptable product with extended shelf-life. HP might increase the safety and protect the product against possible pathogen contamination during processing. © 2011 Elsevier Ltd.

A novel procedure to overcome the seasonal shortage of ovine milk used for cheese manufacture was investigated. Curds made from raw ovine milk in spring months were treated at 400 or 500 MPa of pressure, and frozen for 4 months. After thawing, they were added to fresh bovine curd made from pasteurized milk for the manufacture of experimental Hispánico cheeses. Experimental cheeses exhibited lower dry matter content, less firm texture and higher total free amino acid concentration than a control cheese made from a mixture of pasteurized bovine and ovine milk. Cheese obtained using curds pressurized at 400 MPa showed the highest levels of esterase activity, total free fatty acids, 2-propanol, 2-butanol, 2-pentanol and ethyl hexanoate. The use of frozen pressurized curd made from raw ovine milk for Hispánico cheese manufacture seemed not to have altered its flavour characteristics but increased the yield of the ripe cheese. © 2011 Elsevier Ltd.

In the manufacture of model cheeses, ovine milk was deliberately contaminated with spores of Clostridium beijerinckii INIA 63, a wild isolate from Manchego cheese with late blowing defect, and inoculated with nisin- and lacticin 481-producing Lactococcus lactis subsp. lactis INIA 415 as starter, to test its potential to prevent the late blowing defect, or with L. lactis subsp. lactis INIA 415-2, a spontaneous mutant not producing bacteriocins. Cheeses made individually with the lactococcal strains, without clostridial spores, served as controls. Cheese made with clostridial spores and L. lactis subsp. lactis INIA 415-2 showed late blowing defect after 120. days of ripening. Spoilt cheese also showed lower concentrations of lactic acid, and higher levels of acetic, propionic and butyric acids, and of other volatile compounds such as 2-propanol and 1-butanol, than control cheese. In addition, cheese made with the bacteriocin producer did not show any late blowing symptoms, despite its spore counts similar to those of blown cheese, pointing to outgrowth inhibition of C. beijerinckii spores by bacteriocins. Besides, cheese made with the bacteriocin producer showed similar concentrations of lactic acid and volatile compounds than control cheese. Inclusion of L. lactis subsp. lactis INIA 415 in starter cultures seems a feasible method to prevent late blowing defect in cheese without altering its sensory characteristics. © 2011 Elsevier B.V.

The presence of lactate-fermenting clostridial spores was investigated in 194 samples of raw bulk-tank ovine milk and in 57 Manchego cheeses with late blowing defect collected throughout one year from 55 factories. Spores were detected in 97% of milk samples analysed, with a most probable number (MPN) of 14.5 spores mL-1 and significantly (P < 0.001) higher counts in summer milk. The highest incidence of late blowing defect was also recorded for Manchego cheeses produced in summer. Out of 223 isolates from spoiled cheeses, 78.9%, 10.3%, 9.0% and 1.8% were identified as Clostridium sporogenes, Clostridium beijerinckii, Clostridium tyrobutyricum and Clostridium butyricum, respectively. Among seven bacteriocins produced by lactic acid bacteria, nisin A and Z were the only ones able to inhibit the growth of all Clostridium isolates. © 2010 Elsevier Ltd.

Hispánico cheese is a semi-hard variety, manufactured in Spain from a mixture of pasteurized bovine and ovine milk. To study one strategy for overcoming a seasonal shortage of ovine milk in summer and autumn, curds made from ovine milk, scalded at 32, 35 or 38 °C, were pressed for 30 min and frozen at -24 °C for 4 months. After thawing, they were added to fresh bovine milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized bovine and ovine milk in the same (8020) proportion. No significant effect of the addition of frozen ovine milk curd or scalding temperature was found for lactic acid bacteria counts, dry matter content, hydrophilic and hydrophobic peptides, 45 out of 65 volatile compounds, texture, and sensory characteristics throughout a 60-day ripening period. Differences between cheeses, of low magnitude and little practical significance, were found for pH value, aminopeptidase activity, proteolysis, free amino acids, free fatty acids, and the remaining 20 volatile compounds. Thus, the addition of frozen ovine milk curd to fresh bovine milk curd does not alter the main physicochemical and sensory characteristics of Hispánico cheese. © 2009 Elsevier Ltd. All rights reserved.

Hispánico cheese is manufactured in Spain from a mixture of cow and ewe milk. Production of ewe milk varies throughout the year, with a peak in spring and a valley in summer and autumn. To overcome this seasonal shortage, curd from spring ewe milk may be frozen and used for cheese manufacture some months later. In the present work, ewe milk curds pressed for 15, 60, or 120. min were held at -24°C for 4 mo, thawed, cut to 1-mm pieces, and mixed with fresh cow milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same (8020) proportion. Cheeses, made in duplicate experiments, were analyzed throughout a 60-d ripening period. No significant differences between cheeses were found for lactic acid bacteria counts, dry matter content, hydrophilic peptides, 47 out of 68. vol.tile compounds, texture, and flavor characteristics. On the other hand, differences of minor practical significance between experimental and control cheeses, unrelated to the use of frozen ewe milk curd or the pressing time of ewe milk curd, were found for pH value, aminopeptidase activity, proteolysis, hydrophobic peptides, free amino acids, free fatty acids, and the remaining 21. vol.tile compounds. It may be concluded that the use of frozen ewe milk curd in the manufacture of Hispánico cheese does not alter its main characteristics. © 2010 American Dairy Science Association.

La Serena cheeses, made from Merino ewes' raw milk, were high-pressure (HP)-treated at 300 or 400 MPa for 10 min at 10 °C, on days 2 or 50 of ripening. Cheeses treated by HP on day 2 showed higher pH values than control cheese on day 3, but cheeses treated by HP on days 2 or 50 and control cheese had similar pH values on day 60. Breakdown of caseins was delayed by HP treatment of cheeses on day 2. Cheeses treated by HP on day 2 showed higher levels of hydrophilic peptides, lower levels of hydrophobic peptides, lower hydrophobic peptides hydrophilic peptides ratios, and higher total contents of free amino acids than those of control cheese. HP treatment of cheese on day 50 scarcely affected proteolysis of 60-day-old cheeses. Fracturability, hardness and elasticity values of cheeses treated by HP on day 2 were higher than those of control cheese and of cheeses treated on day 50. Cheeses treated at 400 MPa on day 2 received the lowest scores for quality of taste from panellists, whereas the rest of HP-treated cheeses did not differ from control cheese. © 2007 Elsevier Ltd. All rights reserved.

Lactococcus lactis strain AMP2I expresses OppA(D471R), a mutant oligopeptide binding OppA protein in which the aspartyl residue at position 471 was replaced by arginine. This strain was used as starter culture to investigate its ability to lower the levels of hydrophobic peptides in cheese. A 5-fold increased level of chymosin was used to obtain a high initial level of hydrophobic peptides. Cheese made with L. lactis AMP2I contained lower levels of hydrophobic peptides and showed lower ratios of hydrophobic peptides to hydrophilic peptides than control cheese made with L. lactis NCDO712. Both parameters have been correlated with cheese bitterness, which suggests that bitterness in cheese made with the former strain must be lower than in control cheese. Content of total free amino acids was 1.3-fold higher in cheese made with L. lactis AMP2I than in control cheese, an increase that can be attributed to the expression of the mutant oligopeptide binding OppA protein. © 2006 Elsevier Ltd. All rights reserved.

La Serena cheeses made from raw Merino ewe's milk were high-pressure (HP) treated at 300 or 400 MPa for 10 min on d 2 or 50 after manufacture. Ripening of HPtreated and control cheeses proceeded until d 60 at 8°C. Volatile compounds were determined throughout ripening, and analysis of related sensory characteristics was carried out on ripe cheeses. High-pressure treatments on d 2 enhanced the formation of branched-chain aldehydes and of 2-alcohols except 2-butanol, but retarded that of n-aldehydes, 2-methyl ketones, dihydroxy-ketones, n-alcohols, unsaturated alcohols, ethyl esters, propyl esters, and branched-chain esters. Differences between HP-treated and control cheeses in the levels of some volatile compounds tended to disappear during ripening. The odor of ripe cheeses was scarcely affected by HP treatments on d 2, but aroma quality and intensity scores were lowered in comparison with control cheese of the same age. On the other hand, HP treatments on d 50 did not influence either the volatile compound profile or the sensory characteristics of 60d-old cheese. © American Dairy Science Association, 2007.

Hispánico cheese was manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639, bacteriocin-nonproducing L. lactis ssp. lactis INIA 437, or a combination of both strains, as starter cultures. Lactobacillus helveticus LH 92, a culture of high amino-peptidase activity sensitive to lacticin 481, was added to all vats. Milk inoculation with the bacteriocin producer promoted early lysis of Lb. helveticus cells in cheese. Cell-free aminopeptidase activity in cheese made with the 3 lactic cultures was 1.8 times the level reached in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 15 d of ripening. Proteolysis (as estimated by the o-phthaldialdehyde method) in cheese made with the 3 lactic cultures was twice as high, and the level of total free amino acids 2.4 times the level found in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 25 d of ripening. Hydrophobic and hydrophilic peptides and their ratio were at the lowest levels in cheese made with the 3 lactic cultures, which received the lowest scores for bitterness and the highest scores for taste quality. © American Dairy Science Association, 2006.

La Serena cheese, a Spanish variety made from Merino ewes' raw milk, has a high pH value, low salt content, and high moisture, conditions that are all favorable for growth and survival of contaminating microorganisms, including pathogens. To improve its microbiological quality and safety, high-pressure treatments at 300 or 400 MPa for 10 min at 10°C were applied to 2 batches of La Serena cheese on d 2 or 50 of ripening. Cheese treated on d 2 at 300 MPa showed viable aerobic counts that were 0.99 log units lower than those for control cheese on d 3 and showed counts of enterococci, coagulase-positive staphylococci, gram-negative bacteria, and coliforms that were 2.05, 0.49, 3.14, and 4.13 log units lower, respectively, than control cheese. For cheese treated on d 2 at 400 MPa, the respective reductions in counts were 2.02, 2.68, 1.45, 3.96, and 5.50 log units. On d 60, viable aerobic counts in cheese treated on d 50 at 300 MPa were 0.50 log units lower than those in control cheese, and counts of enterococci, gram-negative bacteria, and coliforms were 1.37, 2.30, and 4.85 log units lower, respectively. For cheese treated on d 50 at 400 MPa, the respective reductions in counts were 1.29, 1.98, 4.47, and >5 log units. High-pressure treatments at 300 or 400 MPa on d 2 or 50 reduced significantly the counts of undesirable microorganisms, improving the microbiological quality and safety of La Serena cheese immediately after treatment and at the end of the ripening period. © American Dairy Science Association, 2006.

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12°C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10°C. Ripening of high-pressure-treated and untreated cheeses continued at 12°C until d 50. High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese. Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity. © American Dairy Science Association, 2006.

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL-1, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g-1 in a control cheese (without BP-LAB, not HP treated), 6.06-6.74 log cfu g-1 in cheeses with BP-LAB, 6.13 log cfu g-1 in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g-1 in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83-5.43 log cfu g-1 in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g-1 or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2. © 2005 Elsevier Ltd. All rights reserved.

The evolution during ripening and the seasonal variation of the volatile fraction of Zamorano cheeses were studied by using purge and trap extraction and GC-MS analysis. Concentrations of acetaldehyde, propanal and branched chain aldehydes, 2-methyl ketones, higher molecular weight alcohols, and most esters increased significantly with ripening time. Branched chain aldehydes were more abundant in spring and winter cheeses. 2-Heptanone showed lower concentrations in autumn cheeses, opposite to 2,3-butanedione and 2,3-pentanedione. Alcohols were in general lower in autumn cheeses, except ethanol and 2-propenol, which were lower in summer cheeses. Methyl-, propyl- and 2-butyl esters were in general less abundant in autumn cheeses. Discriminant analysis classified correctly 90% of the cheeses for the season of manufacture and 100% of the cheeses for the dairy of origin. Principal component analysis showed a positive correlation between odour intensity, aroma intensity, pungent taste and branched chain aldehydes, phenyl acetaldehyde and acetophenone. Odour and aroma qualities were negatively correlated with branched chain alcohols, butyl acetate and 2-butyl acetate. © 2004 Elsevier Ltd. All rights reserved.

Antimicrobial activity of reuterin individually or in combination with nisin against different food-borne Gram-positive and Gram-negative pathogens in milk was investigated. Reuterin (8 AU/ml) exhibited bacteriostatic activity against Listeria monocytogenes, whereas its activity was slightly bactericidal against Staphylococcus aureus at 37°C. Higher bactericidal activity was detected against Escherichia coli O157H7, Salmonella choleraesuis subsp. choleraesuis, Yersinia enterocolitica, Aeromonas hydrophila subsp. hydrophila and Campylobacter jejuni. A significant synergistic effect on L. monocytogenes and a slight additive effect on S. aureus after 24 h at 37°C were observed when reuterin was combined with nisin (100 IU/ml). The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin against Gram-negative pathogens. © 2004 Elsevier B.V. All rights reserved.

Nisin, free or incorporated in calcium alginate microparticles, was added to pasteurized milk (80% cows' and 20% ewes' milk) used for the manufacture of Hispánico cheese with a mesophilic starter and a thermophilic adjunct culture of high aminopeptidase activity. Addition of nisin incorporated in microparticles promoted early lysis of thermophilic adjunct culture bacteria. Extracellular aminopeptidase activity in 1-d-old cheese made with both thermophilic adjunct culture and nisin incorporated in microparticles was 1.8-fold higher than in cheese made with thermophilic adjunct culture and free nisin and 2.0-fold higher than in cheese made only with thermophilic adjunct culture without any addition of nisin. Addition of nisin, free or incorporated in microparticles, did not influence cheese proteolysis measured using hydrophilic or hydrophobic peptides, o-phthaldialdehyde ripening index, or free amino acids. Moreover, a total of 37 volatile compounds were identified in the volatile fraction of Hispánico cheese, using a dynamic headspace technique with a purge and trap system followed by a gas chromatography mass spectrometry analysis. The volatile compound profile was not influenced by nisin addition, either free or incorporated in microparticles, but addition of thermophilic adjunct culture enhanced the formation of 2-butanone, diacetyl, 2,3-pentanedione and acetoin and improved the flavor quality (sensory panel) of cheese.

The production of volatile compounds by wild strains of Lactococcus lactis used as starter cultures and their effect on the sensory characteristics of ewes' raw milk cheese were investigated. Sixteen vats of cheese were manufactured and ripened for 120 d in two experiments, each of them duplicated. In the first experiment, milk was inoculated with different ratios of four wild Lactococcus lactis strains, two producing and two not producing branched-chain volatile compounds, and in the second experiment with different ratios of a commercial starter culture and the two strains producing branched-chain volatile compounds. Cheese pH, proteolysis, and aminopeptidase activity increased when the strains producing branched-chain volatile compounds were inoculated at a higher rate. Fifty volatile compounds were identified in cheeses using a purge and trap system coupled to a gas chromatography-mass spectrometry apparatus. The relative abundances of 30 volatile compounds (8 alcohols, 5 aldehydes, 3 ketones, 12 esters, 1 sulfur compound, and 1 benzenic compound) were influenced by starter culture composition. 2-Methylpropanol, 3-methylbutanol, isobutyl acetate, isoamyl acetate, ethyl butyrate, isobutyl butyrate, and isoamyl butyrate were always more abundant in the cheeses made with a higher level of L. lactis strains producing branched-chain volatile compounds. Flavor intensity was enhanced by a high level of L. lactis strains producing branched-chain volatile compounds in the first experiment, in which four wild L. lactis strains were used as starter culture, but not in the second experiment, in which a combination of two wild L. lactis strains and the commercial starter culture were used. Flavor quality, as judged by trained panelists, was impaired in both experiments by a high level of L. lactis strains producing branched-chain volatile compounds.

Two aminopeptidases, designated as aminopeptidase I (API) and aminopeptidase II (APII), were isolated from the disrupted cells of Brevibacterium linens SR3 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, Q-Sepharose and Sepharose CL-6B chromatography. Optimal temperature for enzymatic activity was 45°C for API and 37°C for APII, and the pH optimum was found to be 8.5 for API and 8.0 for APII. Divalent metals ions like Co2+ and Ni2+ increased enzymatic activity while Ca2+, Cu2+ and Fe2+ had an inhibitory effect. The enzymes were strongly inhibited by EDTA, 1,10-Phenantroline and cysteine, indicating that both aminopeptidases were metalloenzymes. The K(m) values of API and APII for L-leucine-p-nitroanilide were 1.0 and 0.25 mM, respectively. Native molecular masses of aminopeptidases I and II were 80 and 220 kDa after gel filtration chromatography while molecular masses of 14 and 18 kDa, were seen, after SDS-polyacrylamide gel electrophoresis. (C) 2000 Elsevier Science Ltd.

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats' or cows' milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp, lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates. © 2000 Elsevier Science Ltd.

Microbiological, chemical, rheological, and sensory characteristics of Manchego cheese manufactured with raw or pasteurized milk and with a defined-strain starter culture made up of four Lactococcus lactis ssp. lactis isolates from high quality artisanal Manchego cheese have been compared with those of cheese made with a commercial mixed-strain starter culture. Higher total viable counts and lower pH values after 24 h were detected in both raw and pasteurized milk cheeses made with the defined-strain starter culture, which survived better than did the commercial mixed-strain starter culture during ripening of pasteurized milk cheese. Differences in proteolysis of raw milk cheeses were reduced, whereas lower concentrations of residual caseins and higher concentrations of pH 4.6-soluble N and TCA-soluble N were observed in pasteurized milk cheeses made with the commercial mixed-strain starter culture. Elasticity, fracturability, and hardness were higher in raw and pasteurized milk cheeses made with the defined-strain starter culture. Flavor quality and intensity were not apparently affected by the starter culture used, whereas cheese bitterness was lower in raw and in pasteurized milk cheeses made with the defined-strain starter culture.

Chymosin was stable under milk-coagulation and cheese-ripening conditions with a loss of activity not exceeding 8% of the initial level. Cyprosins lost up to 33% of their activity under milk-coagulation conditions and less than 6% under cheese-ripening conditions. Chymosin incubated in the presence of cyprosins lost up to 94% of its activity under milk-coagulation conditions and up to 70% under cheese-ripening conditions.

Raw milk samples from bulk tanks of 114 farms in Central Spain were analysed for Listeria twice per season over a 1-year period. L. monocytogenes and L. innocua were detected in 3.62 and 2.71% of 774 milk samples, respectively. No other Listeria species were isolated. Most farms (85.1%) produced milk apparently free from L. monocytogenes throughout the 1-year sampling period. No seasonal influence on milk contamination by L. monocytogenes or L. innocua was found.

Nisin added at 10 or 100 IU/ml to ultra-high temperature processed (UHT) skim milk had no effect on counts of Listeria monocytogenes after 24 h at 30°C, whereas addition of the lactoperoxidase system (LPS) resulted in counts of viable cells three log units lower than those of control milk after 24 h at 30°C. Addition of nisin and LPS showed a synergistic effect and resulted in counts up to 5.6 log units lower than the control milk. When the two preservatives were added to actively growing cells of L. monocytogenes in two steps with a 2 h interval, their synergistic effect was enhanced. Counts of L. monocytogenes Ohio after 24 h at 30°C in milk with nisin and LPS added together after 3 h of growth were 5.7 log units lower than the control milk. The difference in counts increased to 7.4 log units if LPS was added after 3 h and nisin after 5 h of growth. Similar but less pronounced effects were observed for the more resistant strain L. monocytogenes Scott A.

The inhibitory activity of nisin-producing Lactococcus lactis subsp. lactis ESI 515 on the survival of Listeria innocua during ripening of raw ewes milk Manchego cheese was investigated. After 60 days of ripening, counts of L. innocua in cheese were 4.08 log units lower than the control when Lc. lactis subsp. lactis ESI 515 was used as a single-strain starter. Nisin activity was detected in cheeses manufactured with Lc. lactis subsp. lactis ESI 515 throughout the ripening period.