The accelerating effect of aLactococcus lactis subsp.lactis lactose-utilization/proteinase-deficient strain on proteolysis and flavour development

This study describes the accelerating effect exerted on proteolysis and flavour development in cheese-curd slurries by combined application ofLactococcus lactis subsp.lactis IFPL359 and high concentrations of its Lac^? Prt^? derivative, (i.e. that which has reduced capacity to metabolize lactose and reduced proteolytic activity, strain T1). Cells of strain T1 partially lysed by either sonication or incubation with lysozyme, were also used to ascertain how proteolysis was affected by release of intracellular enzymes in the initial stage of incubation of the strains in cheese-curd slurries. The presence of strain T1 produced higher levels of non-protein nitrogen (NPN) and amine nitrogen (AN) during the first weeks of incubation when partially lysed cells had been added. Addition of whole cells of strain T1 produced higher values of NPN and AN at the end of incubation of slurries, accelerating proteolysis by about 2 weeks with respect to the control, which only contained the parental strain IFPL359. At the end of the experimental period higher amino acid levels were detected by HPLC in the slurry containing whole T1 cells. Volatile fractions of the different cheese-curd slurries were also analysed. The higher level of proteolysis produced by addition of high levels of strain T1 appeared to be related to release of intracellular enzymes by this strain owing to its greater capacity for autolysis.     

Formation of methional by Lactococcus lactis IFPL730 under cheese model conditions

The present work describes the capacity of Lactococcus lactis to produce methional and other sulphur compounds derived from methionine (Met) in a cheese model system. Cheese slurries were prepared from pasteurized ewes' skimmed milk, chemically acidified with glucono-'-lactone and homogenized aseptically adding Met, !-ketoglutarate, pyridoxal 5'-phosphate, thiamine pyrophosphate and NaCl. Slurries were incubated at 12 °C for 14 days with cellular suspensions of L. lactis IFPL359, L. lactis IFPL730 and L. lactis NCDO763 in different combinations. Slurries added with resting cells and the intracellular fraction from L. lactis IFPL730 showed the highest production of methional at the outset of incubation, which decreased during incubation along with a concomitant increase in 3-methylthiopropanol. The sensorial analysis of slurries indicated a characteristic methional aroma (cooked potato-like) in samples containing 4-methylthio-2-ketobutyrate and the intracellular fraction from L. lactis IFPL730. As incubation proceeded, the intensity of methional aroma decreased but samples were judged by the panel tasters as developing a cheese-like flavour.     

Peptidase and proteinase activity ofLactococcus lactis, Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on -casein than on _s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.

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Peptidase- und Proteinase-Aktivität vonLactococcus lactis, Lactobacillus casei undLactobacillus plantarum

Zusammenfassung Das proteolytische System von mehreren nicht kommerziellenLactococcus- undLactobacillus-Stämmen wurde direkt vom traditionellen spanischen halbfesten Ziegenmilchkäse isoliert und untersucht. Die Aktivität von Aminopeptidase, X-Prolyldipeptidylaminopeptidase, Dipeptidase and Proteinase dieser neuen Stämme wurde in cytoplasmatischen, Zellwand-Membran- und spontan freigesetzten Fraktionen gemessen. Die Aminopeptidase-Aktivität erfolgte ausschließlich intracellular und war höher fürLactobacillus casei subsp. casei als fürLactococcus lactis subsp.lactis. Lactobacillus plantarum zeigte höhere Dipeptidase Aktivität alsL. casei. Die höchsten Werte für die Proteinase-Aktivität wurden für die Zellwand-Membran-Fraktion vonLactococcus lactis subsp.lactis IFPL359 gemessen. Für alle untersuchten Stämme war die Aktivität höher bei -Casein als bei _s-Casein. Dieses Ergebnis weist auf den unterschiedlichen Einfluß dieser Stämme bei der Proteolyse von Käse während der Reifung hin. Die Stämme scheinen sich gegenseitig zu ergänzen, wenn sie gemeinsam in Starterkulturen verwendet werden.

     

Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly than _s-cascin.     

Contribution of Lactococcus lactis subsp lactis IFPL 359 and Lactobacillus casei subsp casei IFPL 731 to the Proteolysis of Caprine Curd Slurries

ABSTRACT: Proteolysis was studied in low-fat caprine curd slurries pre-incubated (24 h at 30 °C) with an extra dose of rennet, 10.0 or 22.0 μg g^-1 (LR and HR slurries, respectively), and treated at 70 °C for 15 min prior to incubation with sonicated cells of Lactococcus lactis subsp lactis IFPL 359 alone or combined with Lactobacillus casei subsp casei IFPL 731. The addition of lactobacilli to slurries containing lactococci increased the conversion of water-soluble nitrogen to nonprotein nitrogen and amine nitrogen. Major differences were found for some free amino acids, such as glutamic acid and proline, and hydrolysis of most hydrophobic peptides produced by lactococci, which are associated with bitterness in cheese.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

Caseinolytic activity ofLactococcus lactis subsp. lactis: study of different culture media

Summary Different caseinolytic activity levels ofLactococcus lactis subsp.lactis IFPL 367 were obtained depending upon the culture medium tested. Activity on the-casein fraction was in all cases higher than activity on other casein fractions. The highest activity level was recorded for bacterial cells cultured in a casein-based medium that also contained 0.1% yeast extract, 20 mmol/CaCl₂, and 1% lactose. The effect of the components of the growth media on the proteolytic activity of the bacterial strain employed in the experiment is also discussed.

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Caseinolytische Aktivität vonLactococcus lactis subsp. lactis: Einfluß des Nachweismediums

Zusammenfassung Die verschiedene caseinolytische Aktivität ist von dem Nachweismedium abhängig. Die-Casein-Aktivität der Kultur war in jedem Fall größer als die Aktivität der anderen Casein-Fraktionen. Die höchste Aktivität wurde beim Wachstum in einem Casein-Nährboden mit 0,1% Hefeextrakt, 20 mmol CaCl₂ und 1% Lactose gefunden. Der Einfluß des Nährbodens auf die proteolytische Aktivität der untersuchten Keime wurde ebenfalls ermittelt.

     

Use of a bacteriocin-producing transconjugant as starter in acceleration of cheese ripening.

The non-conjugative 46 kb plasmid that encodes the biosynthesis of lacticin 3147 in Lactococcus lactis IFPL105 has been transferred to the starter L. lactis IFPL359, used in goat's milk cheesemaking. The accelerating effect exerted on proteolysis and development of sensory characteristics of semi-hard cheese by the bacteriocin-producing transconjugant L. lactis IFPL3593 (Lac+ Bac+ Imm+), which is able to induce cell lysis in starter adjuncts with high peptidase activity, has been studied. It has been demonstrated that the use of IFPL3593 as starter accelerates cheese ripening as it increases the level of amino nitrogen correlated with early cell lysis of adjuncts. The fact that the bacteriocin-producing microorganism used is immune to the bacteriocin. allowed proper acidification of the curd without altering the cheese-making process.     

Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly thanα _s-cascin.     

Control of late blowing in cheese by adding lacticin 3147-producing Lactococcus lactis IFPL 3593 to the starter

Bacteriocins produced by lactic acid bacteria offer potential as tools for ensuring food safety and quality. Lacticin 3147 is a two-peptide lantibiotic previously shown to inhibit a broad range of food spoilage and pathogenic bacteria. Lacticin 3147-producing Lactococcus lactis IFPL 3593 was investigated for its ability to inhibit the growth of clostridia as a means to prevent late blowing in cheese. L. lactis IFPL 3593 was shown to inhibit germination of clostridia spores and prevent late blowing in semi-hard cheeses, with a 5 log g^?1 reduction in the numbers of spores when compared to control cheeses. Furthermore, this bacteriocin-producing strain demonstrated considerable potential as a biopreservative agent against heterofermentative lactobacilli and their associated blowing defects. The use of this strain to prevent late blowing in cheese thereby represents a promising alternative to the addition of lysozyme particularly given the increasing concerns regarding the potential allergenicity of this additive.     

Cooperation between wild lactococcal strains for cheese aroma formation

Several wild lactococcal strains were tested for their ability to produce aroma compounds during growth in milk. Strains were incubated alone and in combination with Lactococcus lactis IFPL730, which is characterized by showing α-keto acid decarboxylase activity. Volatile compounds from incubated milks were analyzed by means of solid-phase microextraction (SPME) and evaluated by gas chromatography–mass spectrometry (GC–MS). Incubated milks were also sniffed for sensory analysis to describe aroma attributes. The combination of L. lactis IFPL326 that showed the highest branched chain aminotransferase activity with IFPL730 contributed to the highest formation of leucine-derived volatile compounds, such as 3-methylbutanal, 3-methyl-1-butanol and 2-hydroxy-4-methyl pentanoic acid methyl ester. In addition, the milk incubated with this combination of strains was awarded, by the test panellists, the highest scores for “ripened cheese” attribute and aroma intensity. The results indicate that combination of L. lactis strains harbouring complementary catabolic routes can contribute to improved cheese aroma formation, the combined cultures with L. lactis IFPL730 resulting in higher volatile compound formation than isolate strains.     

Proteolytic properties of Turkish white‐brined cheese (Beyaz peynir) made by using wild‐type Lactococcal strains

The development of proteolysis in white‐brined Turkish cheese made by using wild strains of Lactococcus lactis subsp. lactis (namely MBLL9, MBLL23 and MBL27) was monitored for 90 days. Proteolysis in cheeses was investigated using urea‐PAGE gel electrophoresis of pH 4.6‐insoluble and RP‐HPLC of both 70% ethanol‐insoluble and 70% ethanol‐soluble nitrogen fractions. Results indicated that developments of proteolysis in the experimental cheeses were strain dependent. The degradation of casein fractions was more evident in the cheeses made using strain MBLL23. The lowest levels of proteolysis and development of acidity were obtained in the cheese made using strain MBLL9.     

Influence of a bacteriocin-producing lactic culture on proteolysis and texture of Hispánico cheese

Hispánico cheese was manufactured in duplicate experiments, each consisting of two 50-L vats, and ripened for 75 days. Lactic cultures for experimental cheese were 0.5% Lactococcus lactis subsp. lactis INIA 415 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain), a bacteriocin-producing (Bac⁺) strain harbouring the structural genes of nisin Z and lacticin 481, 0.5% L. lactis subsp. lactis INIA 415-2, a Bac⁻ mutant and 2% TA052, a commercial Streptococcus thermophilus culture. Lactic cultures for control cheese were 1% L. lactis subsp. lactis INIA 415-2 and 2% TA052. S. thermophilus counts were lower, and levels of cell-free intracellular aminopeptidases higher, from day 1 in cheese made with the bacteriocin producer, indicating early lysis of the thermophilic culture. Experimental cheese showed reduced proteolysis of α_s-casein and lower levels of hydrophilic and hydrophobic peptides than control cheese. However, proteolysis as estimated by the o-phthaldialdehyde method and total level of free amino acids were in experimental cheese 1.38- and 2.47-fold, respectively, those in control cheese on day 25, and 1.49- and 2.34-fold, respectively, on day 75. Higher values of fracturability, elasticity and hardness were recorded from day 50 for cheese made with the bacteriocin producer, which were related to its higher residual α_s-casein content. The use of a bacteriocin-producing culture, though retarding α_s-casein proteolysis and softening of texture, enhanced considerably secondary proteolysis during cheese ripening.     

Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins

A non-toxigenic strain of proteolytic Penicillium chrysogenum Pg222 isolated from dry-cured ham was tested for its ability to generate non-protein nitrogen (NPN) and volatile compounds from muscle myofibrillar proteins. The activity of mold led to higher accumulation of non-protein and amino acidic nitrogens than control samples. Volatile compound analysis revealed the presence of branched compounds, such as 3-methylbutanal and 3-methylbutanol only in samples inoculated with the mold. Similarly, compounds such as ethanol, propanol and 2-methoxy ethanol were detected only in inoculated samples at all sampling time. 3- and 2-methylpentane, benzoic and acetic acids, 2-butanone and 2-ethylhexanol, pyridine and 3-carene were detected occasionally, but only in the Pg222 batch. The proteolytic activity of P. chrysogenum Pg222 lead to accumulation of soluble NPN compounds, in addition to the generation of volatile compounds of great interest for dry-cured meat products. Therefore, this mold could be appropriate to be used as a non-toxigenic starter culture during the ripening of dry-cured meat products to stimulate proteolysis and flavour development.     

Proteolysis in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ŁOCK 0900 probiotic strain

Selected parameters related to proteolysis were evaluated in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ?OCK 0900 probiotic strain. Dehydration, pH, water activity and total nitrogen content were not affected by ultrasound treatment. Neither sonication nor inoculation has an influence on L* and a* colour parameters. Sonication significantly influenced the pattern of proteolysis providing higher nonprotein nitrogen content (NPN) and proteolysis index (PI). The highest intensity of proteolysis, as assessed by NPN and PI, occurred in an inoculated sample. Neither sonication nor inoculation with L. casei had a significant effect on total viable counts. Loins inoculated with L. casei had the highest lactic acid bacteria count, followed by the sonicated and nonsonicated control. This research shows that sonication followed by inoculation with L. casei ?OCK 0900 could be used as an effective measure to speed up the proteolytic changes in dry‐aged meat cuts.     

Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins

Probiotic cells were microencapsulated in milk protein matrices by means of an enzymatic induced gelation with rennet. Water insoluble, spherical capsules with a volume-based median of 68 ± 5 μm were obtained from a novel developed emulsifying and subsequent internal gelation process. A high encapsulation yield was found due to the encapsulation procedure for Lactobacillus paracasei ssp. paracasei F19 and Bifidobacterium lactis Bb12. After incubation at low pH-values, microencapsulation yielded higher survival rates compared to non-encapsulated probiotic cells. The viable cell numbers of encapsulated Lactobacillus paracasei and Bifidobacterium lactis were 0.8 and 2.8 log units CFU g⁻¹ higher compared to free cells after 90 min incubation at pH 2.5. The improved survival of encapsulated cells can probably be explained by a higher local pH-value within the protein matrix of the capsules caused by the protein buffering capacity, protecting the cells during incubation under simulated gastric conditions at low pH. The study indicates that rennet-induced gelation of skim-milk concentrates for the microencapsulation of probiotic cells can be a suitable alternative to current available technologies, mainly based on ionotrophic gelation of plant-polymer solutions.     

Effect of pre-ripening on microbial and chemical changes in dry fermented sausages

The effect of pre-ripening at 5°C on microbial changes, nitrate reduction and proteolysis in both naturally fermented and inoculated (Lactobacillus sakeandStaphylococcus carnosus) sausages was investigated.Micrococcaceaelevels were significantly higher (P<0.05) in pre-ripened batches, whether or not starter cultures were inoculated; this was in accordance with both the lower (P<0.05) lactic acid bacteria levels and higher (P<0.05) pH values reached at the end of the process. In non-inoculated batches contents of residual nitrate were lower (P<0.05) as a consequence of the stronger (P<0.05) pH drop caused byL. sakeinoculation which can inhibit nitrate reductase activity. However, this inhibitory effect can be significantly (P<0.05) reduced by applying a pre-ripening treatment. Maximal non-protein nitrogen (NPN) and amine nitrogen (AN) increases were detected in inoculated batches submitted to pre-ripening, although a general significant (P<0.05) effect of pre-ripening on proteolysis was not inferred. Pre-ripening can therefore be considered as a way of preventing an earlyMicrococcaceaeinhibition and achieving a more effective nitrate reduction when inoculating a mixed starter culture including a lactic acid bacteria strain.     

Fresh-cheesemilk formulation fermented by a combination of freeze-dried citrate-positive cultures and exopolysaccharide-producing lactobacilli with liquid lactococcal starters

Milk formulation (4% fat and 5% protein) prepared to simulate fresh cheese production was inoculated with: (1) 10⁷ cfu mL⁻¹ of fresh liquid starters of Lactococcus lactis ssp. lactis T1 and Lc. lactis ssp. cremoris T2, (2) a freeze-dried exopolysaccharide-producing (EPS) strain of Lactobacillus rhamnosus RW-9595M, and (3) freeze-dried Leuconostoc cremoris LM057 or Lc. lactis ssp. lactis var. diacetylactis MD089 strains. The effect of inoculation rate of the freeze-dried starters (between 10⁶ and 10⁷ cfu mL⁻¹) and incubation temperature (between 23.5 and 36.5 °C) on evolution of pH and the various populations during fermentation was examined. Texture (apparent viscosity, syneresis potential) and chemical composition (diacetyl, acetaldehyde) of the fermented milks were also determined. Milk was incubated until a pH of 4.6 was obtained, which required between 6 and 10 h depending on temperature.In the range of inoculation levels used, there was no significant effect of the presence of lactobacilli, Ln. cremoris or Lc. lactis ssp. lactis var. diacetilactis on the growth of the lactococci. There was a direct correlation between the inoculation rates of the freeze-dried cultures and their final populations in the fermented milks. The growth of the cultures were also affected by temperature, Ln. cremoris growing less as incubation temperature increased, while the opposite was noted with Lb. rhamnosus. The apparent viscosity of the fermented milk was significantly affected by incubation temperature, but there was no correlation between apparent viscosity and the final population in lactobacilli. Of the three variables studied, the highest correlation with diacetyl content was obtained with the inoculation level of the Leuconostoc strain.     

Expression of amino acid converting enzymes and production of volatile compounds by Lactococcus lactis IFPL953

Lactococcus lactis contains a large number of key enzymes responsible for the formation of volatile compounds characteristic of cheese flavour. In the present work we have investigated the expression of genes codifying amino acid converting enzymes (AACE) and the formation of volatile compounds by L. lactis IFPL953 and its mutant lacking the hydroxy acid dehydrogenase PanE (L. lactis IFPL953ΔpanE). The growth in absence of isoleucine was the main induction factor in the expression of araT, bcaT, panE, and kivD. The expression of the gdh gene of L. lactis IFPL953ΔpanE increased remarkably during the stationary growth phase, particularly under isoleucine and valine starvation conditions. L. lactis IFPL953ΔpanE showed an enhanced formation of 2- and 3-methylbutanal and their corresponding alcohols and an increase in the formation of ketones. These findings contribute to a better knowledge of L. lactis AACE regulation and its potential application in cheese flavour formation.