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Dynamics of probiotics Lactobacillus rhamnosus LGG, L. reuteri BioGaia, Bifidobacterium animalis subsp. lactis BB12 and B. longum subsp. longum BB536 were investigated during the fermentation of milk supplemented with extracts of Chondrus crispus (CC), Himanthalia elongata (HE), Laminaria ochroleuca (LO), Palmaria palmata (PP), Porphyra umbilicalis (PU), Ulva lactuca (UL) and Undaria pinnatifida (UP). After 24 h at 37 °C, LGG attained counts above 108 cfu/ml in control milk and in milk with all seaweed extracts while BioGaia only attained that level in milk with CC, HE, PP, PU and UL extracts, B12 in milk with UL and UP extracts, and BB536 in milk with CC, HE, PU and UL extracts. In physiological saline, LGG, BioGaia, BB12 and BB536 were respectively stimulated by 4, 0, 5 and 3 seaweed extracts after 24 h at 37 °C and inhibited by 1, 5, 2 and 0 seaweed extracts. Casein and whey proteins added to physiological saline neutralized inhibition caused by UL extract, increasing probiotic counts by 4–5 log cfu/ml, while lactose and citrate had no significant effect. Milk supplementation with seaweed extracts permitted attaining probiotic counts above 108 cfu/ml at the end of fermentation, a result of practical interest for the dairy industry.

The effect on the biochemical characteristics of Iberico 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.

Supplementation of dairy products with seaweeds can contribute to attaining a balanced diet, but they may affect their sensory characteristics. Yogurt and quark were supplemented with dehydrated Himanthalia elongata, Porphyra umbilicalis, Saccharina latissima, Ulva lactuca and Undaria pinnatifida. When odour, flavour and texture were evaluated by panellists, dairy products containing 0.50% U. pinnatifida showed the highest seaweed flavour and the lowest flavour quality, while dairy products containing 0.50% S. latissima showed the lowest seaweed flavour and the highest flavour quality. Seaweed species influenced all yogurt attributes except buttery odour, acid flavour and salty flavour and all quark attributes except yogurt odour, acid flavour and sweet flavour. Logarithmic regressions of seaweed odour and flavour on seaweed concentration (0–1.00%) were the best fit for dairy products containing P. umbilicalis, U. lactuca or U. pinnatifida and linear regressions for those supplemented with H. elongata or S. latissima. © 2016 Institute of Food Science and Technology

Kombu seaweed (Laminaria ochroleuca) was preserved by refrigeration at 5 °C (control), by high pressure processing (HPP) at 400 or 600 MPa for 5 min followed by refrigeration at 5 °C, or by freezing at −24 °C. Those treatments were applied to both unsalted and salted kombu samples, which were stored for 180 days. One hundred three compounds belonging to 16 chemical groups were identified in the volatile fraction of kombu. Groups including the highest number of volatile compounds were alcohols (24), aldehydes (13), ketones (10), alkanes (9), haloalkanes (9), alkenes (8) and acids (7). HPP lowered the levels of acids, aldehydes, ketones, lactones + ionones, pyranones and terpenes + terpenoids, and increased alkenes and furans. Freezing lowered the levels of alkanes, aromatic hydrocarbons, pyranones and terpenes + terpenoids, and increased aldehydes, haloalkanes and thiazoles. Salting lowered the levels of pyranones, and increased acids, alcohols, aldehydes, alkanes, aromatic hydrocarbons, furans, haloalkanes, lactones + ionones and thiazoles. Control kombu odour acceptance fell below rejection level on day 60 while HPP-treated kombu showed the highest odour acceptance until day 180. Frozen kombu exhibited abnormal odours whereas salted kombu maintained acceptable odour characteristics. Principal component analysis associated the most relevant groups of volatile compounds with kombu microbiological and sensory characteristics.

[Background] Lactococcus lactis is the main component of the mesophilic starters used in cheese manufacture. The success of milk fermentation relies on the viability and metabolic activity of the starter bacteria. Therefore, robust strains able to withstand the harsh conditions encountered during cheese manufacture and starter production are demanded. In this work, we have applied adaptive evolution under cell envelope stress imposed by the cell wall active bacteriocin Lcn972 to evolve strains with more robust phenotypes., [Results] Consecutive exposure of the starter strain L. lactis IPLA947 to Lcn972 yielded a stable mutant, L. lactis R5, with enhanced survival when challenged with hydrogen peroxide. L. lactis R5 exhibited faster growth rates in aerobic fermentations in broth and was able to acidify milk to a lower pH in aerated milk cultures. The improved behavior of L. lactis R5 in the presence of oxygen did not translate into a better performance in the presence of heme (i.e. respiration metabolism) or into higher survival during storage at cold temperatures or after freeze-drying compared to the wild type L. lactis IPLA947. L. lactis R5 retained the same milk acidification rate and no changes in the consumption of lactose and production of organic acids were noticed. However, the profile of volatile compounds revealed a significant increase in 3-hydroxy-2-butanone (acetoin) in curds manufactured with L. lactis R5., [Conclusions] Based on our results, L. lactis R5 can be proposed as a suitable dairy starter with improved survival under oxidative stress and enhanced metabolic traits. The results support the notion that adaptive evolution under cell envelope stress might be useful to generate strain diversity within industrial L. lactis strains., This research was supported by grant BIO2013–46266-R and BIO2017–88147-R (Ministerio de Economía y Competitividad, Spain). Activities of the DairySafe group at IPLA-CSIC were also funded by GRUPIN14–139 (FEDER funds and program of Science, Technology and Innovation 2013–2017, Principado de Asturias, Spain).

The microbiota of eight species (Chondrus crispus, Himanthalia elongata, Laminaria ochroleuca, Palmaria palmata, Porphyra umbilicalis, Saccharina latissima, Ulva lactuca and Undaria pinnatifida) of edible seaweeds collected in North West Spain, marketed as dehydrated product, was quantitatively determined on nine solid media. Representative colonies were selected from solid culture media. The isolated microorganisms were identified by means of morphological characteristics, 16S rDNA sequencing and biochemical tests. U. pinnatifida was the seaweed species showing the most abundant microbial population, with counts on Marine agar up to 7.7 log cfu/g in individual samples and 5.0 log cfu/g as the mean value, and counts of coliforms up to 4.6 log cfu/g in individual samples and 2.4 log cfu/g as the mean value. The 225 identified bacterial isolates belonged to 11 families, 27 genera and 56 species. Bacillaceae was the family accounting for the highest number of isolates (111) followed by Enterobacteriaceae (60), Bacillales Family XII Incertae Sedis (20), Planococcaceae (11), Moraxellaceae (7), Paenibacillaceae (5) and Pseudomonadaceae (5). Bacterial species showing the highest occurrence in dehydrated seaweeds were Bacillus megaterium, B. licheniformis, Pantoea sp. and termoresistant Pantoea sp. Four of the Bacillus species isolated from dehydrated seaweeds (B. cereus, B. licheniformis, B. pumilus and B. subtilis) are among those containing strains considered to be foodborne pathogens and nine of the isolated non-Bacillales bacterial species have been reported to contain human opportunistic pathogenic strains.

The effect of high pressure processing (HPP) at 600 MPa and refrigerated storage for 5 months on the volatile fraction of 30 Iberian dry-cured hams of different compositional characteristics was investigated. Compositional characteristics significantly influenced 11 compounds (3 alcohols, 3 carboxylic acids, 2 alkanes, 2 benzene compounds and 1 aldehyde) out of the 116 compounds identified in the volatile fraction of Iberian ham. HPP treatment had a significant effect on 34 volatile compounds, with higher levels of 11 compounds and lower levels of 23 compounds in HPP-treated samples than in control samples. Refrigerated storage for 5 months significantly influenced the levels of 75 compounds, 25 of which appeared or increased while 50 disappeared or decreased. During that period, the total abundance of volatile compounds decreased by 5.1% in control ham and 1.3% in HPP-treated ham. Iberian ham commercial shelf life should be limited to maintain its sensory characteristics. Industrial relevance HPP treatment and refrigerated storage of dry-cured ham are advantageous industrial and commercial procedures. In the particular case of Iberian ham, both HPP and refrigerated storage brought about a decrease in the total levels of volatile compounds. Therefore, adequate HPP treatment conditions and commercial shelf life should be cautiously set in order to safeguard the unique odor and aroma characteristics of Iberian ham.

Supplementation of dairy products with non-dairy ingredients with the aim of increasing the concentration of particular nutrients is a common industrial practice. In the present study, a semi-hard cheese was supplemented with each of five seaweeds (Himanthalia elongata, Laminaria ochroleuca, Porphyra umbilicalis, Ulva lactuca, Undaria pinnatifida). Addition of 10 g of dehydrated seaweed per kg of curd caused significant differences in dry matter and pH value, but it hardly influenced cheese microbiota. Antioxidant activity, which was correlated with total phenolic compounds, was significantly higher in cheese supplemented with H. elongata than in control cheese. Instrumental colour and texture parameters of cheese varied significantly with the added seaweed species. Cheeses supplemented with H. elongata, U. pinnatifida and L. ochroleuca received odour and flavour quality scores from panellists that did not differ from those of the respective control cheese until day 60 and were associated with low seaweed odour and flavour scores.

The volatile fraction of 30 Iberian dry-cured hams of different physicochemical characteristics and the effect of high pressure processing (HPP) at 600 MPa on volatile compounds were investigated. According to the analysis of variance carried out on the levels of 122 volatile compounds, intramuscular fat content influenced the levels of 8 benzene compounds, 5 carboxylic acids, 2 ketones, 2 furanones, 1 alcohol, 1 aldehyde and 1 sulfur compound, salt concentration influenced the levels of 1 aldehyde and 1 ketone, salt-in-lean ratio had no effect on volatile compounds, and water activity influenced the levels of 3 sulfur compounds, 1 alcohol and 1 aldehyde. HPP-treated samples of Iberian ham had higher levels of 4 compounds and lower levels of 31 compounds than untreated samples. A higher influence of HPP treatment on volatile compounds than physicochemical characteristics was observed for Iberian ham. Therefore, HPP treatment conditions should be optimized in order to diminish its possible effect on Iberian ham odor and aroma characteristics. © 2017 Elsevier Ltd

The volatile fraction of dehydrated edible seaweeds belonging to seven species (Himanthalia elongata, Laminaria ochroleuca, Palmaria palmata, Porphyra umbilicalis, Saccharina latissima, Ulva lactuca and Undaria pinnatifida) was analyzed by gas chromatography–mass spectrometry, after solid-phase microextraction of samples. Thirty-six hydrocarbons, 34 ketones, 28 aldehydes, 23 alcohols, 8 carboxylic acids, 6 halogenated compounds, 4 furans, 3 esters, 2 sulphur compounds, 2 pyrazines, 1 pyridine and 1 amine were detected among the 151 volatile compounds found in seaweeds. There were significant differences between seaweed species for all the volatile compounds. Hydrocarbons reached their highest levels in U. pinnatifida, ketones in P. umbilicalis, aldehydes in P. palmata and P. umbilicalis, alcohols in P. umbilicalis, carboxylic acids in S. latissima, and halogenated compounds in L. ochroleuca and S. latissima. Sensory analysis revealed that P. palmata, U. lactuca and H. elongata were the seaweeds showing the most potent seafood odour and seaweed odour characteristics. © 2016 Elsevier Ltd

The microbiota of Serrano dry-cured ham of different chemical composition, subjected or not to high-pressure processing (HPP), was investigated using culture-dependent and culture-independent methods. Microbial counts were submitted to analysis of variance with physicochemical parameters (a w , NaCl concentration, salt-in-lean ratio and intramuscular fat content) or HPP as main effects. In untreated hams, physicochemical parameters significantly affected counts of aerobic mesophiles, psychrotrophs, and moulds and yeasts. NaCl concentration and fat content influenced the levels of four and three of the five studied microbial groups, respectively, whereas no influence of a w was stated. The HPP treatment had a significant effect on counts of all investigated microbial groups. Culture-independent methods showed the presence of bacteria such as Staphylococcus equorum , Staphylococcus succinus , Bacillus subtilis and Cellulosimicrobium sp., moulds like Penicillium commune , Aspergillus fumigatus , Sclerotinia sclerotiorum , Eurotium athecium and Moniliella mellis , and yeasts like Debaryomyces hansenii and Candida glucosophila . Absence of B . subtilis bands and weaker bands of E . athecium were recorded for HPP-treated hams. The higher microbial levels found in lean ham might result in a quicker deterioration. HPP treatment confirmed its suitability as a procedure to control spoilage microorganisms. DGGE did not seem to be sensitive enough to highlight changes caused by HPP treatment in the microbiota of ham, but contributed to the detection of microbial species not previously found in ham.

Cheese varieties with long ripening periods are prone to form biogenic amines and develop off-flavours. High-pressure processing (HPP), which inactivates microorganisms and enzymes, may be useful in preventing those defects. On this aim, cheeses made from unpasteurized milk were treated at 400 or 600 MPa, after 14 or 21 days of ripening, and their characteristics were compared to those of untreated control cheese throughout a 240-day period. Lactic acid bacteria declined by 2 log units in 400 MPa cheeses and by 6 log units in 600 MPa cheeses after HPP, while Gram-negative bacteria were below detection level in all the HPP-treated cheeses. Aminopeptidase activity was significantly lower in HPP cheeses than in control cheese from day 21 onwards. Hydrolysis of αs-casein was enhanced in 400 MPa cheeses, but not in 600 MPa cheeses, while a more pronounced hydrolysis of β-, κ- and para-κ-caseins was recorded in all the HPP cheeses from day 60 onwards. Levels of hydrophilic and hydrophobic peptides were higher in HPP cheeses than in control cheese on day 60 and thereafter. Total free amino acids were at lower concentrations in 600 MPa cheeses than in the rest from day 60 onwards. The concentration of total biogenic amines was lower in all the HPP cheeses than in control cheese from day 60 onwards. Flavour quality and flavour intensity of cheese made from unpasteurized milk were not significantly affected by HPP. © 2014, Springer Science+Business Media New York.

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.

One hundred and three volatile compounds were detected by solid-phase microextraction followed by gas chromatography–mass spectrometry in 30 ripened Serrano dry-cured hams, submitted or not to high pressure processing (HPP) and afterwards held for 5 months at 4 °C. The effect of ham physicochemical parameters and HPP (600 MPa for 6 min) on volatile compounds was assessed. Physicochemical parameters primarily affected the levels of acids, alcohols, alkanes, esters, benzene compounds, sulfur compounds and some miscellaneous compounds. Intramuscular fat content was the physicochemical parameter with the most pronounced effect on the volatile fraction of untreated Serrano ham after refrigerated storage, influencing the levels of 38 volatile compounds while aw, salt content and salt-in-lean ratio respectively influenced the levels of 4, 4 and 5 volatile compounds. HPP treatment affected 21 volatile compounds, resulting in higher levels of alkanes and ketones and lower levels of esters and secondary alcohols, what might affect Serrano ham odor and aroma after 5 months of refrigerated storage. © 2016 Elsevier Ltd

Raw goat milk collected at 11 Andalusian dairy plants was used to individually manufacture cheeses without starter culture addition. Microbial groups were monitored during ripening for 60 days. A total of 1442 lactic acid bacteria (LAB) isolates randomly chosen were identified by molecular methods. Counts of mesophilic LAB exceeded 9 log cfu g-1 on day 1 and decreased 0.5 log units during ripening whereas counts of lactobacilli and leuconostocs increased and those of enterococci remained constant. Thirty-three LAB species belonging to eight different genera were isolated, with higher biodiversity on day 1 (26 species) than on day 60 (20 species). Structural genes coding for nisin, lacticin 481, lactococcin B, six plantaricins and five enterocins were detected in LAB isolates. The information obtained on the technological properties of selected isolates may be useful in developing specific starters for goat milk cheeses, capable of maintaining their distinctive sensory characteristics. © 2015 Elsevier Ltd.

Hispánico cheese, manufactured from a mixture of cow and ewe milk, is representative of cheese varieties made using milk from more than one animal species in Mediterranean countries. The shortage of ewe milk production in autumn hinders the uniformity of Hispánico cheese composition throughout the year. To surmount this inconvenience of ewe milk seasonality, curds made in spring from raw and pasteurized ewe milk were stored frozen and used four months later for the manufacture of Hispánico cheese. Experimental cheeses were made by mixing fresh curd from pasteurized cow milk with thawed curd from raw or pasteurized ewe milk, and control cheese from a mixture of pasteurized cow and ewe milk in the same proportion. Characteristics of experimental and control cheeses throughout a 60-d ripening period were investigated. On the one hand, the experimental cheese containing frozen curd from raw ewe milk showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, the highest levels of aminopeptidase and esterase activity, and the highest concentrations of free amino acids, free fatty acids, alcohols and esters. On the other, the experimental cheese containing frozen curd from pasteurized ewe milk had concentrations of free amino acids, free fatty acids and volatile compounds similar to those of control cheese, with the only exception being a higher level of ketones. Flavour intensity reached the highest scores in the experimental cheese containing frozen curd from raw ewe milk, followed by the experimental cheese containing frozen curd from pasteurized ewe milk. Flavour quality scores of both experimental cheeses were similar, and lower than those of control cheese. Copyright © Proprietors of Journal of Dairy Research 2012.

High-pressure (HP) treatment of curd at low temperatures, on top of efficiently reducing microbial loads while maintaining most of the enzymatic activity, can be useful to overcome the seasonal shortage in ovine milk production. In this work, raw ovine milk curds were subjected to a wide range of HP treatments and, after frozen storage, used in the manufacture of Hispánico cheese. Confocal scanning laser microscopy of HP-treated curds showed increasing denser and more compact protein networks with the pressure applied. Although no differences in overall porosity were recorded between HP-treated and non-pressurised curds, HP treatment lowered pore number and modified pore size and shape. Whereas confocal scanning laser microscopy of control cheese showed open structures with numerous and irregular cavities, additional highly compact protein network areas could be observed in experimental cheeses micrographs. These differences trailed off with time and were no longer observed after 60 days of ripening. Experimental cheeses had significantly lower dry matter values than control cheese, and higher proteolytic indexes. Their texture was less firm than that of control cheese throughout the whole ripening period, requiring less force to be broken and deformed. Experimental cheeses showed lower lightness values than control cheese after 30 days of ripening, but no differences were found after 60 days. Our results indicate that frozen storage of HP-treated curds from ovine milk is a viable option for the production of Hispánico cheese. © 2012 Springer Science+Business Media, LLC.

High-pressure treatment of raw goat milk curd was investigated as an alternative to thermal treatment of milk in cheese manufacture, and curd freezing as a procedure to surmount the seasonality of goat milk production. Experimental cheeses were made by mixing (7030) fresh cow milk curd with frozen curd from pasteurized goat milk (PGC), frozen curd from raw goat milk (RGC), or frozen pressurized curd from raw goat milk (PRGC). Control cheese was made from a mixture (7030) of pasteurized cow and goat milk. RGC cheese showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, whereas PRGC cheese had maximum aminopeptidase activity, esterase activity, and overall proteolysis. Control cheese exhibited the highest dry matter content and peptide levels, the lowest concentration of free amino acids, the highest concentration of volatile compounds such as free fatty acids, alcohols and esters, and the firmest texture. Differences in sensory characteristics between experimental and control cheeses were of minor importance. High-pressure treatment of curd allowed the production of cheese of bacteriological quality similar to that of control cheese made using pasteurized milk, while curd freezing did not alter the sensory characteristics of experimental cheeses with respect to control cheese. © 2012 Springer Science+Business Media, LLC.

Lactococcus lactisstrain AMP2I expresses OppA(D471R), a mutant oligopeptide binding OppA protein in which the aspartyl residue at position 471 was replaced by arginine. As a consequence of a different peptide transport in this strain, experimental Hispánico cheese made withLc. lactisAMP2I had a higher content of total free amino acids than control cheese made withLc. lactisAMP1I, an isogenic strain expressing wild-type OppA (Picon et al. 2005, 2007). In this work higher levels of diketones, hydroxy-ketones and, to a lesser extent, branched chain aldehydes were recorded for experimental cheese compared with control cheese. These differences levelled off as ripening proceeded. Strong correlations support the hypothesis that the increased levels of these volatile compounds in cheese made withLc. lactisAMP2I are linked to higher concentrations of free amino acids threonine, valine and leucine.

Cheeses made from unpasteurized milk were high pressure processed (HPP) for 14 or 21 days after manufacture at 400 or 600 MPa, in order to prevent excessive accumulation of flavour compounds, in particular free fatty acids (FFA) and volatile compounds (VC). They were compared with untreated control cheese throughout a 240-day period. On day 60, cheeses treated at 400 MPa had esterase activity values 57.1–58.0 % lower than control cheese while cheeses treated at 600 MPa had values 82.3–82.8 % lower, with no significant differences between total FFA concentrations of cheeses. However, on day 240, total FFA concentrations were 6.5–9.0 % lower in 400 MPa cheeses than in control cheese and 15.0–16.9 % lower in 600 MPa cheeses, with more marked differences for short-chain FFA and less marked for hexadecanoic and octadecanoic acids. Fifty-two of the 70 VC found in cheese were significantly influenced by HPP. On day 240, total alcohols reached the highest levels in control and 400 MPa cheeses, total aldehydes, ketones and hydrocarbons in 600 MPa cheeses, and total sulphur compounds in 400 and 600 MPa cheeses. Levels of total volatile acids, esters, ethers, benzenic compounds and terpenoids in 240-day cheese were not affected by HPP. Odour quality and intensity were not significantly influenced by HPP. Higher L* values were recorded for 600 MPa cheeses and higher a* values for control and 400 MPa cheeses. Principal component analysis on the groups of volatile compounds highlighted the different evolvement of volatiles in control, 400 and 600 MPa cheeses during ripening. © 2015, Springer Science+Business Media New York.

Lactic starters used for cheese manufacture play an important role in the production of bitter peptides and their degradation to non-bitter products. The oligopeptide transport system (Opp) of lactococci is essential for milk peptide utilization. The periplasmic substrate binding protein serves to capture the substrate with high affinity and to deliver it to a membrane-bound complex that translocates it inside the cell. Prt+- and Lac+- derivatives of MG1363 ΔoppA strains expressing a wild-type MG1363 OppA or a mutant OppA with a single point mutation at residue 471 (OppA(D471R)) from a plasmid were constructed. These strains were used as lactic starters in cheese manufacture to improve flavour quality by removing hydrophobic peptides from the cheese matrix, through their preferential transport by OppA(D471R). Cheeses made with these strains were not significantly different from control cheeses after 1 day of ripening with respect to bacterial counts, pH and proteolysis, and only slight differences were recorded after 9 and 20 days of ripening. HPLC chromatograms of the hydrophilic and hydrophobic peptides present in the water-soluble fraction of experimental cheeses showed significant differences in peptide content as well as in peak profiles. These results suggest a different peptide utilization in the strain expressing OppA(D471R) and make it suitable for use as starter to improve cheese quality. © 2005 Elsevier B.V. All rights reserved.

Although glucose is an inexpensive substrate widely used as a carbon source in Escherichia coli recombinant fermentation technology, 10-30% of the carbon supply is wasted by excreting acetate. In addition to the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. Because glucose can enter the cell via several transport systems, isogenic strains defective in one or two of these transport systems were constructed. The effects of changes in the glucose uptake capacity on the in vivo flux distribution to a desired end product (β-galactosidase) and to acetate were studied. The lack of one of the components (IICB Glc protein) of the glucose-phosphoenol-pyruvate phosphotransferase system (Glc-PTS) reduced the growth rate significantly. The maintenance of a low-copy plasmid in this strain resulted in further arrest of the growth rate. However, β-galactosidase production had no effect on growth rate. This strain directed more carbon into biomass and carbon dioxide, and less into acetate, β-Galactosidase was produced in amounts not significantly different from the wild-type strain from half the amount of glucose. An explanation for the experimental results is given, making use of published results on metabolic regulation. © 2005 Wiley Periodicals, Inc.

Brie cheeses were high pressure (HP)-treated at 400 or 600MPa, on days 14 or 21 after manufacture, to prevent over-ripening. HP-treatment reduced total free fatty acid content of 120-day-old cheese by up to 88.5%. On day 120, HP-treated cheeses had levels of alcohols, aldehydes, acids, esters and ethers up to 3.4, 1.9, 43.8, 18.7 and 5.6 times higher, respectively, than control cheese, while ketones and hydrocarbons levels were up to 88.6% and 48.9% lower, respectively. Levels of sulphur compounds, pyrazines and amines increased drastically in control cheese from day 60 onwards, resulting in lower odour quality scores. On day 120, HP-treated cheeses had levels of sulphur compounds, pyrazines and amines up to 96.9%, 99.3% and 99.4% lower, respectively, than control cheese. Cheese appearance was impaired by HP-treatment, resulting in lower lightness and slightly more reddish and yellowish colour. These changes might diminish consumer acceptance of HP-treated Brie cheese. © 2014 Elsevier Ltd.

Build-up of flavour compounds throughout ripening of raw milk cheeses may result in strong over-ripening notes during refrigerated storage. In order to control the formation of free fatty acids (FFAs) and volatile compounds, and the appearance of off-odours, raw milk cheeses were high-pressure-processed (HPP) 21 or 35 days after manufacture at 400 or 600 MPa. Ripening proceeded at 8 °C until day 60 and, afterwards, cheeses were held at 4 °C until day 240. The effect of HPP on the formation of FFAs and volatile compounds was dependent on pressure level and cheese age at the time of treatment. On day 60, acetic and propionic acids, branched-chain FFAs and short-chain FFAs showed the lowest (p < 0.05) concentrations in cheeses treated at 400 or 600 MPa on day 21, while medium- and long-chain FFAs were at similar levels in all cheeses. HPP influenced significantly (p < 0.05) 84 out of the 94 volatile compounds found in cheese. On day 60, the lowest (p < 0.05) concentrations of acids, alcohols and esters were recorded for cheeses treated at 400 or 600 MPa on day 21, and the lowest (p < 0.05) concentrations of ketones for cheeses treated at 400 MPa on days 21 or 35. On day 240, all HPP cheeses showed lower (p < 0.05) concentrations of aldehydes, esters and, particularly, sulphur compounds than control cheese, which exhibited putrid and rancid off-odours from day 120 onwards. Principal component analysis combining FFAs and volatile compounds discriminated 240-day control cheese from 120-day control cheese and both from the rest of cheeses. © 2013 Springer Science+Business Media New York.

High-pressure-processing (HPP) at 400 or 600 MPa was applied to cheeses made from ewe raw milk, on days 21 or 35 after manufacturing, to reduce proteolysis and prevent over-ripening. The characteristics of HPP and non-pressurized (control) cheeses were compared during ripening at 8 °C until day 60 and further storage at 4 °C until day 240. HPP and control cheeses showed similar pH values throughout ripening, but on day 240 pH values remained 0.4-0.6 units lower for HPP cheeses than for the control cheeses. Casein degradation was significantly retarded in the 600 MPa cheeses. Their α-casein concentration was 48-52 % higher on day 60 and 30-33 % higher on day 240 than in the control cheeses while β-casein concentration was 25-26 % higher on day 60 and 100-103 % higher on day 240. No significant differences in para-κ-casein concentration between cheeses were found on day 60, but on day 240, it was 22-35 % higher in the 600 MPa cheeses than in the control cheese. Hydrophilic peptides, hydrophobic peptides and total free amino acids evolved similarly in HPP and control cheeses during the 60-day ripening period. However, on day 240 hydrophilic peptides were at 34-39 % lower levels in the 600 MPa cheeses than in the control cheeses, hydrophobic peptides at 7-16 % lower levels and total free amino acids at 25-29 % lower levels. Flavour intensity scores increased at a slower rate in HPP cheeses than in the control cheese. Flavour quality declined markedly in the control cheeses during refrigerated storage while it did not vary significantly in 600 MPa cheeses. © 2013 Springer Science+Business Media New York.

The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis , taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar K m for transport, whereas the V max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters. This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues. Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand. The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides. This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA. Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants. These results are discussed in relation to the affinity constant for transport of these substrates. Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.

Chymosin, a neutral proteinase from Bacillus subtilis and cardoon cyprosins, were co-encapsulated with phospholipase C in stimulated release liposomes. Encapsulated enzymes were added separately to milk to make cheese. Chymosin and the neutral proteinase accelerated as-casein degradation in comparison with control cheese, whereas b-casein degradation was accelerated by neutral proteinase and cyprosins. Neutral proteinase yielded the highest increase in soluble nitrogen. Cheese flavour intensity was enhanced by the neutral proteinase and cyprosins but not by chymosin.

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 applied to HPP cheeses. © 2013 American Dairy Science Association.

Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids.

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600. MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400. MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600. MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600. MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600. MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600. MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics. © 2013 American Dairy Science Association.

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.

To overcome the seasonal shortage of goat milk in mixed milk cheese manufacture, pasteurized goat milk curd and high-pressure-treated raw goat milk curd manufactured in the spring were held at -24°C for 4 mo, thawed, and mixed with fresh cow milk curd for the manufacture of experimental cheeses. Control cheeses were made from a mixture of pasteurized cow and goat milk. The microbiota of experimental and control cheeses was studied using culture-dependent and culture-independent techniques. Bacterial enumeration by classical methods showed lactic acid bacteria to be the dominant population in both control and experimental cheeses. In total, 681 isolates were grouped by partial amplified rDNA restriction analysis (ARDRA) into 4 groups and identified by 16S rRNA gene sequencing as Lactococcus lactis ssp. lactis (563 isolates), Leuconostoc pseudomesenteroides (72 isolates), Lactobacillus spp. (34 isolates), and Lc. lactis ssp. cremoris (12 isolates). Temporal temperature gradient gel electrophoresis (TTGE) analysis of cheese showed (1) the predominance of Lc. lactis in all cheeses; (2) the presence of Leu. pseudomesenteroides population in all cheeses from d 15 onward; (3) the presence of a Lactobacillus plantarum population in control cheese until d 15 and in experimental cheeses throughout the ripening period. Due to the most diverse and complete set of peptidases present in the genus Lactobacillus, the prevalence of this population in experimental cheeses could give rise to differences in cheese flavor between experimental and control cheeses. © 2011 American Dairy Science Association.

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.

The increasing demand for tasty dairy products has raised the interest for strains of lactic acid bacteria with novel properties. In this study we have explored the proteolytic system of 24 wild Lactococcus lactis strains isolated previously from Spanish raw milk cheeses, to select proteolytic strains with high peptidase activity and efficient peptide transport capability. Large variations in overall proteolysis, proteolytic activities, as well as in peptide utilization, were recorded among strains. Peptide utilization correlated well with the presence of oligopeptide transport systems (Opp and Dpp) in wild L. lactis strains. Eighteen of the 24 wild L. lactis strains possessed both Opp and Dpp systems, what supports the theory that the presence of both oligopeptide transport systems could confer an advantage to the strain. Differences in the nucleotide sequence of genes coding for the oligopeptide binding proteins were shown by means of restriction endonuclease analyses. Based on the characteristics determined in this work, L. lactis strains ESI197, M21 and P21 seem to be promising candidates for use as components of starter cultures. © 2009 Elsevier Ltd. All rights reserved.

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.

During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate.

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 106 to 107 cfu mL- 1 after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P < 0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R). © 2007 Elsevier B.V. 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.

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.

Cyprosins, the proteinases present in cardoon rennet, were extracted from dried flowers of cardoon ( Cynara cardunculus L.) and encapsulated in dehydration-rehydration liposomes. Liposomes were added to pasteurized ovine milk to accelerate the ripening of Manchego cheese. Encapsulated proteinases had no effect on whey composition, but DM and protein content were lower in 1-d-old experimental cheese than in the respective control cheese. Enhancement of proteolysis by encapsulated cyprosins was evident 24h after manufacture; 38.3% of α s -CN and 47.7% of β -CN that were initially present in milk had been degraded in l-d-old experimental cheese and 20.3 and 37.5%, respectively, in control cheese. At that time, amounts of N that were soluble at pH 4.6, in TCA, and in phosphotungstic acid were 15.70, 5.47, and 2.09% in experimental cheese and 9.32, 4.16, and 1.34% in control cheese, respectively. The hydrophobic and hydrophilic peptides that were separated by HPLC and measured at 214 and 280nm, and their ratio, were also higher in experimental cheese than in control cheese on d 1. However, some of the indices of proteolysis were higher in control cheese than in experimental cheese after 60 d of ripening. Experimental cheese was softer than control cheese 24h after manufacture, but texture was not different thereafter. Addition of encapsulated cyprosins to milk perceptibly accelerated development of flavor intensity in experimental cheese through 15 d of age without enhancing bitterness.

Bacillus subtilis neutral proteinase was encapsulated in conventional dehydration-rehydration liposomes and, co-encapsulated with a phospholipase, in triggered liposomes. Triggered liposomes had released in buffer 58.0% of the encapsulated proteinase after 180 min at 30°C, whereas only 1.1% was released from conventional liposomes. Addition of triggered liposomes to milk resulted in only 70.4% residual β-casein after 240 min at 30°C compared to 90.7% in control milk. If triggered liposomes were added to milk with lactic starter and rennet, residual β-casein was only 52.3% after 240 min, compared to 80.3% in milk without liposomes. © 1995 Chapman & Hall.

Neutral proteinase from Bacillus subtilis was entrapped in dehydration rehydration liposomes, which were added to pasteurized ewe milk to accelerate Manchego cheese ripening; proteinase level was, 040 Anson units/kg in fresh curd. Encapsulated proteinase had no apparent effect on whey composition or on dry cheese yield, but experimental cheese showed a higher DM from d 15 and a higher protein content from d 30. Proteolysis was enhanced by encapsulated proteinase by 24 h after manufacture; 26.5% of αs1-casein and 53.4% of β-casein that was initially present in milk had been degraded in 1-d-old control cheese and 51.3 and 83.5%, respectively, in experimental cheese. After 90 d of ripening, amounts of N that were soluble at pH 4.6, in TCA, and in phosphotungstic acid were 34.09, 17.50, and 11.88% in experimental cheese and 20.09, 13.67, and 8.58% in control cheese, respectively. Hydrophobic and hydrophilic peptides measured at 214 and 280 nm were higher in experimental cheese than in control cheese from d 15. Higher fracturability, elasticity, and hardness values were recorded for experimental cheese despite its more extensive proteolysis; the firmer texture was ascribed to its higher DM content. No bitter flavor defect was detected in experimental cheese, although development of flavor intensity was accelerated by encapsulated proteinase. © 1995, American Dairy Science Association. All rights reserved.

Kinetics of milk coagulation by three proteolytic enzymes, chymosin, bovine pepsin and neutrase, at varying concentrations, were monitored by thrombelastography. Clotting time for milk coagulation by chymosin was linear (r2= 0.997) with the reciprocal of enzyme concentration, whereas the best equation for pepsin (r2 = 0.997) and neutrase (r2 = 0.999) was the one relating the reciprocal of clotting time to enzyme concentration. When the effect of mixtures of enzymes on milk coagulation was studied, the best-fitting regression was that of the reciprocal of clotting time on concentration of enzymes for both chymosin-pepsin (r2 = 0.998) and chymosin-neutrase (r2 = 0.998) mixtures.

The effect of liposome-encapsulated chymosin derived by fermentation (Kluyveromyces lactis) on Manchego cheese characteristics was investigated. Encapsulation efficiency was 12.6% when the enzyme was entrapped in phospholipid liposomes prepared by the dehydration-rehydration procedure. Addition of liposome-encapsulated enzyme to pasteurized ewe milk in Manchego cheese making increased chymosin derived by fermentation 2.6-fold in experimental curd compared with control curd and enhanced degradation of αs1- and ß-caseins during cheese ripening. After 60 d of maturation, amounts of N soluble at pH 4.6, in TCA, and in phosphotungstic acid were 16.66, 9.48, and 3.79% in experimental cheese and 14.43, 8.31, and 3.19% in control cheese, respectively. Cheese texture in experimental vats softened because of the more extensive proteolysis. Concentration of hydrophobic peptides, flavor preference, flavor intensity, and bitterness score were significantly influenced by cheese age, but not by the addition of liposome-encapsulated enzyme. © 1994, American Dairy Science Association. All rights reserved.

Cyprosin extract from dried flowers of Cynara cardunculus L. was dissolved in two different buffers (50 mM Tris-HCl, pH 8.3 and 10 mM PBS, pH 7.4), mixed with a sonicated soybean lecithin dispersion and microencapsulated in dehydration-rehydration liposomes. Efficiency of cyprosin encapsulation was 12.1% for Tris-HCl liposomes and 12.3% for PBS liposomes. TCA-soluble N in 24 h cheese was higher when PBS liposomes were added to milk (A280=0.604) than when Tris-HCl liposomes (A280=0.392) or no liposomes (A280=0.359) were added, due to the efficient delivery of cyprosin into the curd by PBS liposomes. © 1994 Kluwer Academic Publishers.

Cellophane and polyethylene packaging in conjunction with precooling, a modified atmosphere and an ethylene absorber were tested in order to obtain a more extended postharvest life of strawberry Chandler and Douglas cultivars. Treatment had a significant influence on all the physical and chemical characteristics investigated. Low density polyethylene bags with or without a CO 2 atmosphere resulted in the lowest weight losses, conductivity and degree of fruit decay, together with the highest firmness values. The use of an ethylene absorber constituted an additional cost without a proven benefit.

An accurate and objective thrombelastographic procedure for the quantitative determination of chymosin in solution and in cheese whey is proposed. Linear relationships between coagulation time and the inverse of chymosin concentration in solution showed determination coefficients (r2) in the range 0·989-0·997. A satisfactory reproducibility was achieved, with 0·88% as mean coefficient of variation. Determination coefficients for linear relationships between coagulation time and the inverse of chymosin present in whey ranged from 0·995 to 0·998. Reproducibility of chymosin quantification in whey was high, with 0·98% as mean coefficient of variation. © 1993.