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In this study, a collection of 298 wild strains of lactic acid bacteria (LAB) isolated from raw goat milk cheeses, belonging to 8 genera and 24 species, was investigated for their ability to generate volatile compounds. Sensory evaluation showed that goat milk cultures of Leuconostoc and Lactococcus strains reached the highest scores for dairy odour attributes. Fifty six LAB strains with the highest sensory evaluation scores (one per each ten, or fraction, of those belonging to the same species) were selected for gas chromatography–mass spectrometry (GC–MS) analysis. A total of 34 volatile compounds (five carboxylic acids, eleven alcohols, six aldehydes, six ketones, one ester, and five miscellaneous compounds) were detected in lactic curds made with each one of 56 selected LAB strains. The number of volatile compounds in individual curds varied from 15 to 33 while the total abundance of volatile compounds in individual curds ranged from 1.98- to 17.50-fold the total abundance in uninoculated curd. Major differences in volatile generation were related to amino acid catabolism.

[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).

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.

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.

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.

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.

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.

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.

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.