Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

Influence on cheese proteolysis and sensory characteristics of non-starter lactobacilli strains with probiotic potential

The contribution to cheese proteolysis and sensory profile of four potentially probiotic non-starter lactobacilli strains was assessed in two different models: soft and semi-hard cheeses. All the strains were able to grow in both types of cheese, where they maintained high levels during ripening. Overall, adjunct cultures of Lactobacillus rhamnosus showed the strongest influence in peptidolysis, which was verified by changes in peptide profiles and increase of free amino acids concentration. Nevertheless, some cheeses treated with L. rhamnosus also showed post-acidification during ripening and decreased sensory characteristics compared to controls. Cheeses with adjunct cultures of Lactobacillus casei I90 and Lactobacillus plantarum I91 exhibited, in general, an intermediate level of peptidolysis between control and L. rhamnosus-added cheeses; their sensory characteristics were preserved or improved, and they did not show any defects. Adjunct cultures showed similar trends in both models, confirming L. casei I90 and L. plantarum I91 as the most performing adjunct cultures for cheese-making among the strains.     

Microbiological,compositional, biochemical and textural characterisation of Caciocavallo Pugliese cheese during ripening

A microbiological, compositional, biochemical and textural characterisation of the pasta filata Caciocavallo Pugliese cheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g⁻¹ mesophilic bacteria and ca. log 6.0 cfu g⁻¹ presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum, Lb. gasseri, Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of α_s1-casein was more rapid than that of β-casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of Caciocavallo Pugliese cheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese.     

Polyphasic characterization of indigenous lactobacilli and lactococci from PDO Canestrato Pugliese cheese

A polyphasic approach, involving both genotypic and phenotypic analyses was used to characterize 33 isolates of lactic acid bacteria from the raw milk Protected Designation of Origin (PDO) Canestrato Pugliese cheese, in order to select candidate strains that can be used as autochthonous starter cultures in the dairy industry. Randomly amplified polymorphic DNA (RAPD) analysis and clustering by the unweighted pair-group method with arithmetic averages (UPGMA) were used to evaluate the genotypic diversity, while phenotypic characterization was performed through miniaturized assays and traditional biochemical tests. Technological properties of major interest for cheese-making (acidification, tendency to lysis, proteolytic and peptidase activity) were also evaluated, and selected subset of data was statistically examined by the one-way ANOVA and the Tukey-Kramer HSD test, to highlight strain-specific and species-specific differences among the isolates. A high degree of diversity appeared in the phenotypic and technological traits in opposition to a relatively low genotypic diversity. Although none of the isolates showed the best performances in all the activities, an appropriate mixture of strains could be selected for providing an efficacious autochthonous starter culture.     

Contribution of Geotrichum candidum to the proteolysis of soft cheese

To determine the action of the yeast Geotrichum candidum on the proteolysis of soft cheese, Camembert-type cheeses were manufactured with and without this surface flora. Casein degradation and the release of peptides and amino acids at the cheese surface were studied to assess overall proteolysis. The results showed extensive proteolytic activity at the surface of cheese with G. candidum, and suggested that G. candidum is able to contribute to both primary and secondary proteolysis. α_s1- and βA²-caseins were preferentially hydrolysed at the surface of cheese with G. candidum from the first week of ripening. This proteolytic activity led to the production of numerous peptides that were subsequently hydrolysed, as indicated by the large increase in the concentration of free amino acids from the second week to the end of ripening.     

Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening.To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed.We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively.The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.     

Effects of genetic type,stage of lactation,and ripening time on Caciocavallo cheese proteolysis

The objective of this experiment was to evaluate the effects of genetic type, stage of lactation, and ripening time on proteolysis in Caciocavallo cheese. One hundred twenty Caciocavallo cheeses made from the milk of 2 breeds, Italian Brown and Italian Holstein and characterized by different stages of lactation were obtained and ripened for 1, 30, 60, 90, and 150 d. Cheese proteolysis was investigated by ripening index (ratio of water-soluble N at pH 4.6 to total protein, %) and by the study of degradation of the protein fractions (α_S1-, β-, and para-κ-casein), which was determined by densitometric analysis of isoelectric focusing results. The statistical analysis showed a significant effect of the studied factors. Ripening index was higher in Italian Brown Caciocavallo cheese and in cheeses made with early lactation milk, whereas casein solubilization was greater in the first 2 mo of ripening. Isoelectric focusing analysis of cheese samples during ripening showed extensive hydrolysis of caseins. In particular, the protein fraction that underwent major degradation by proteolytic enzymes was α_S1-casein, followed by β-casein, whereas para-κ-casein was less degraded. Italian Brown cheese showed a lower residual quantity of β- and para-κ-casein, whereas Italian Holstein cheese showed a lower residual quantity of α_S1-casein. In addition, significant interactions of both first and second order were found on both ripening index and degradation of protein fractions. This study demonstrated that the analyzed factors influenced proteolysis of Caciocavallo cheese, which forms the basis of new knowledge that could lead to the production of a pasta filata cheese with specific characteristics.     

Methods used to study non-starter microorganisms in cheese: a review

The significance of non-starter lactic acid bacteria (NSLAB), which become the dominant organ-isms in hard cheese varieties during ripening, is still controversial, but they have been shown to contribute to the release of amino acids and to influence flavour development. Since the NSLAB population is essentially uncontrolled, it is likely that at least some of the variability in cheese quality is due to the NSLAB. Several approaches used to assess the significance of NSLAB in cheese ripening are reviewed in this paper.     

Textural changes of Canestrello Pugliese cheese measured during storage

The changes in the fundamental textural properties of the Canestrello Pugliese cheese, during ripening, were studied, accounting for the internal and external portions of the cheese. The cheese was ripened at 12–14 °C and 80% relative humidity and analyzed at 0, 15, 30, 45, 60, 95, and 120 days to determine the moisture content, water activity (A_w), pH values and some fundamental-mechanical and dynamic-mechanical properties such as the elastic modulus (E_c), storage modulus (G′), loss modulus (G″) and relaxation times distribution curve. Significant differences in the moisture content, A_w, pH and the elastic modulus were observed for the inside and outside portions of the investigated cheese. In particular, for the first 60 days of ripening the elastic modulus of the outer region was higher than that of the inner region. As the ripening process approached the end of the fourth month the contrary was true. Moreover, results show that, above all the pH and A_w play a major role in determining the mechanical properties of the Canestrello Pugliese cheese during ripening.     

Consumer acceptance and sensory evaluation of Monti Dauni Meridionali Caciocavallo cheese

Twelve Caciocavallo cheeses were collected from 6 factories (A, B, C, D, E, F) located in the Monti Dauni Meridionali area (Southern Italy) that adopted different protocols for cheese production. A total of 160 consumers were involved in the sensory evaluation of Caciocavallo cheese after 180 d of ripening. Cheese attributes were used to describe the flavor, texture, and appearance of cheeses. The highest scores for the shiny attribute were assigned to cheeses B, C, and E, whereas color intensity was the highest in cheeses B, D, and F. Strength, salty, and piquant attributes were higher in cheeses F and A because of the use of raw milk (F), rennet paste (A), and percentage of salt in the brine (A, F). Consumers perceived a more granular structure during the second half of chewing of Caciocavallo cheese F, as evidenced by the highest value for the grainy attribute. A positive correlation was found between overall flavor and odor intensity and water-soluble nitrogen, low molecular weight peptides, and free fatty acids and between piquant and butyric and caproic acids. A principal components analysis applied to the sensory attributes accounted for 65% of the total variance. The score plot showed that cheeses F and A were located in a well-defined zone of the plot, with cheeses in this zone displaying higher levels of strength, piquant, and salty attributes. The preference test assigned 40% of the preference to Caciocavallo cheese A, 38% to cheese F, 9% to cheese E, 8% to cheese D, and 7% to cheeses B and C. Sensory evaluation of Monti Dauni Meridionali Caciocavallo cheeses is a useful analysis to highlight the principal attributes able to influence consumers' liking that are related to biochemical features of the cheese.     

干酪加工中的牛奶内源酶和非发酵剂乳酸菌

介绍了干酪加工中起作用的牛奶内源酶及微生物，包括血浆酶、组织蛋白酶D、半胱氨酸蛋白酶、脂蛋白脂肪酶和非发酵剂乳酸菌及它们的作用机制和特性。     

Technological and probiotic role of adjunct cultures of non-starter lactobacilli in soft cheeses

The influence of two cheese-isolated Lactobacillus strains on cheese composition, acceptability and probiotic capacity was assessed. Soft cheeses with and without the addition of Lactobacillus plantarum I91 or Lactobacillus paracasei I90 were prepared. Gross composition was assessed and secondary proteolysis was described by soluble fractions and free amino acids profiles. Acceptability was determined by a panel of 98 non-trained consumers. Cheeses harboring added Lactobacillus strains were also studied in vivo to evaluate their probiotic capacity. Gross composition of the cheeses was similar for control and treated (Lactobacillus-added) cheeses. Peptidolysis increased in cheeses with added lactobacilli, which was evidenced by a higher free amino acid content. Overall, the acceptability of the cheeses was good: 65%–80% of the consumers said that they “liked very much” or “liked” the cheeses. Cheeses with L. plantarum I91 showed the highest changes in composition and proteolysis and were the most accepted ones. On the contrary, composition of cheeses with L. paracasei I90 was similar to that of the controls, but these samples were less accepted than cheeses without lactobacilli. The oral administration of cheese containing L. plantarum I91 or L. paracasei I90 proved to be safe and able to enhance the number of IgA + cells in the small intestine lamina propria of mice. The use of selected strains of NSLAB exerted a technological and probiotic role: it contributed to the standardization of cheese quality and induced benefic health effects at the gut mucosa in vivo.     

Use of mass spectrometry to characterize proteolysis in cheese

A rapid method for characterizing proteolysis in different cheese varieties was developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). pH 4.6-soluble extracts of different cheese varieties were sub-fractionated using ethanol and 70% ethanol-soluble extracts were analyzed using MALDI-ToF-MS. Sample analysis time was ∼3 min compared to ∼100 min for reversed-phase HPLC; moreover, the method yielded accurate molecular masses of peptides originating as a result of proteolysis. Small peptides (<3500 m/z) were monitored and data for relative intensities of peptides were analyzed using multidimensional scaling (MDS) to demonstrate the similarity between profiles obtained from different cheese varieties. The similarities between the profiles of MALDI-ToF mass spectra were demonstrated in a two-dimensional space by performing MDS on the similarity matrix. Use of MALDI-ToF-MS is thus a fast and effective method for monitoring small peptides produced in cheese as a result of proteolysis.     

Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese

The range of carbon sources available in cheese curd during maturation that could be used as energy and growth substrates by 60 cultures of non-starter lactic acid bacteria isolated from Cheddar cheese was determined by the detection of tetrazolium salt reduction in Biolog MT1 microplates^™. There were marked inter-species and strain differences in the range of carbon substrates catabolized by the 11 Lactobacillus spp. and 2 Weissella spp. examined. Sugars were used widely among the NSLAB with 90, 100 and 85% of the isolates metabolizing lactose, glucose and galactose, respectively. In addition, ribose, N-acetyl-galactosamine and sialic acid, potentially derived from nucleic acid and casein deglycosylation, were catabolized by 58, 48 and 22% of the isolates, respectively. Lactic acid was also a potential substrate for 15% of the isolates but Tween 80 was not an effective substrate. Although 50% of the NSLAB removed citric acid from the growth medium it was not an independent energy source. Peptides and amino acids were also catabolized by up to 27% of the NSLAB provided that an exogenous source of α-ketoglutaric acid was present to facilitate the aminotransferase-mediated transamination degradative pathway. The MT1 microplate method facilitates the rapid screening for isolates able to establish in the cheese curd and for the detection of specific metabolic activities in isolates undergoing evaluation for use as adjunct cultures in cheesemaking trials.     

Survival of some non-starter bacteria in naturally ripened and enzyme-accelerated Cheddar cheese

Effects of the addition of a proteinase (Neutrase 1-5S) and a peptidase (aminopeptidase DP-102) as agents for accelerating the ripening of Cheddar cheese on the survival of some non-starter bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and a Salmonella sp.) were studied throughout a 4-month ripening period. The enzymes were found to have no significant effect on the survival of the Gram-positive bacteria but some significant effects were observed, at some stages of the ripening period, with the Gram-negative bacteria in that lower levels were recovered from cheeses treated with the enzyme system.     

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4.6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of alpha- and beta-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.     

Evaluation of genetic polymorphism among Lactobacillus rhamnosus non-starter Parmigiano Reggiano cheese strains

Parmigiano Reggiano (PR) is an Italian cooked, long-ripened cheese made with unheated cow's milk and natural whey starter. The microflora is involved in the manufacturing of this cheese, arising from the natural whey starter, the raw milk and the environment. Molecular studies have shown that mesophilic non-starter lactic acid bacteria (NSLAB) are the dominant microflora present during the ripening of PR. In this study, a characterisation of Lactobacillus rhamnosus isolated from a single PR manufacturing and ripening process is reported, using a combination of genotypic fingerprinting techniques (RAPD-PCR and REP-PCR). The intraspecies heterogeneity evidenced for 66 strains is correlated to their abilities to adapt to specific environmental and technological conditions. The detection of biotypes that correlate with specific moments in cheese ripening or differential development throughout this process suggests that these strains may have specific roles closely linked to their peculiar technological properties.     

Intervarietal comparison of proteolysis in commercial cheese

 Proteolysis in different varieties of cheese, i.e. Cheddar, British, Dutch and Swiss types and Italian varieties, was compared by polyacrylamide gel electrophoresis (PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). Urea-PAGE of the water-insoluble fraction (WISF) of cheese appeared to be unable to distinguish between Cheddar and Dutch-type cheeses, whereas it could be used to distinguish Emmental from Parmesan and both of these from Cheddar and Dutch types. Urea-PAGE of the 70%-ethanol-insoluble fraction of the water-soluble extract (WSE) showed large inter-varietal differences but there were also some intra-varietal differences. RP-HPLC of the 70%-ethanol-soluble and -insoluble fractions of the WSEs of cheeses was more effective than urea-PAGE of the WISFs or of the 70%-ethanol-insoluble fraction of the WSEs of cheeses when classifying cheese according to variety. However, analysis of a larger number of cheese samples is required to verify this result. One of the problems with using either urea-PAGE or RP-HPLC to identify cheese is that the characteristics analysed by these techniques change as ripening progresses. Received: 22 September 1997 / Revised version: 8 June 1998     

Fermentation of carbohydrates from cheese sources by non-starter lactic acid bacteria isolated from semi-hard Danish cheese

Carbohydrate fermentation of 45 isolates of non-starter lactic acid bacteria from Danish semi-hard cheeses was studied using BioScreen C equipment. Thirty-nine of the isolates were identified as Lactobacillus paracasei/casei/rhamnosus, 2 as Lb. curvatus and 4 as a new species, Lb. danicus, using ITS-PCR. A specially designed carbohydrate-restricted medium supplemented with one of nine carbohydrates was used to evaluate potential carbohydrate sources in cheese–milk-fat globule membrane (MFGM), glycomacropeptide (GMP), or lysed cells. Lb. paracasei strains grew well on the carbohydrates from MFGM, GMP and bacterial cell wall peptidoglycan. The highest growth rates were observed on N-acetylglucosamine (NAG) (0.32–0.56 h⁻¹) and the lowest on ribose (0.12–0.23 h⁻¹, if ribose was fermented at all). Lb. danicus strains grew better on carbohydrates from lysed bacterial cells than on carbohydrates from MFGM or GMP, and it was the only species with a shorter lag-phase on ribose or NAG after being pre-incubated on ribose.