Modulation of casein proteolysis by lactococcal peptidase gene inactivation

Peptidases from lactic acid bacteria participate in cheese ripening by hydrolysing peptides and liberating free amino acids, which are precursors of aroma compounds. Using a pseudo-curd model and lactococcal mutants, negative for different peptidases, as ripening agents, we determined some of the key peptidases in the ripening process. The total level of amino acids quantified after 28 days of ripening was significantly reduced when the general aminopeptidase PepN was absent. The situation was aggravated when the tripeptidase PepT was also missing. The deficiency in the proline-specific aminopeptidase PepX more specifically decreased the level of free proline in the pseudo-curds after 28 days of ripening. These three peptidases, which are also necessary for optimal lactococcal growth in milk, can be considered as peptidases of technological importance. On the other hand, the suppression of the other peptidases tested (PepF1, PepF2, PepC, PepP) did not significantly affect amino acid pools in the conditions of the test.     

Characterization of aromatic properties of old-style cheese starters

Old-style cheese starters were evaluated to determine their ability to produce cheese aroma compounds. Detailed analyses of the aroma-producing potential of 13 old-style starter cultures were undertaken. The proteolytic profile of the starters was established by an accelerated ripening study using a model cheese slurry and compared with those of a commercial aromatic starter and commercial Cheddar cheeses. To evaluate the aromatic potential of the starter cultures, quantification of free amino acids liberated and volatile compounds after 15 d of ripening at 30°C as well as sensory analysis were carried out. Results showed that proteolysis patterns of all 13 starter cultures in the curd model were comparable to those of commercial Cheddar cheeses. All tested cultures demonstrated the ability to produce high amounts of amino acids recognized as precursors of aroma compounds. Several differences were observed between the starters and commercial Cheddar cheeses regarding some amino acids such as glutamate, leucine, phenylalanine, proline, and ornithine, reflecting the various enzymatic systems present in the starters. Starters Bt (control) and ULAAC-E exhibited various significant differences regarding their free amino acid profiles, as confirmed by sensory analysis. In addition, identification of volatile compounds confirmed the presence of several key molecules related to aroma, such as 3-methylbutanal and diacetyl. Besides the aroma-producing aspect, 2 starters (ULAAC-A and ULAAC-H) seem to possess an important ability to generate large amounts of γ-aminobutyric acid, which contributed up to 15% of the total amino acids present in the model curd after 15 d ripening. γ-Aminobutyric acid is an amine well-known for its antihypertensive and calming effects.     

Effect of heterofermentative lactic acid bacteria of DL-starters in initial ripening of semi-hard cheese

The role of heterofermentative lactic acid bacteria from dl-starters in ripening of semi-hard cheese was investigated using the strains Leuconostoc pseudomesenteroides PS12 and 1159, Leuconostoc mesenteroides subsp. cremoris T26 and Lactobacillus danicus 13M1. Control cheese was made with starter containing only homofermentative Lactococcus lactis subspecies. Leuc. mesenteroides subsp. cremoris T26 did not grow in cheese and started to decrease in number early, whereas the others grew and remained at a high number throughout the nine-week ripening period. None of the added heterofermentative strains affected proteolysis and total amount of amino acids; however, differences in the composition of amino acids were observed, and caused significant differences in the composition of volatile aroma compounds. Added strains increased the amount of secondary alcohols and mediated decreases in the amount of corresponding methyl ketones, diacetyl and acetoin. Eye formation was only affected by Lb. danicus through stimulation of late gas formation in cheeses.     

Two strains of nonstarter lactobacilli increased the production of flavor compounds in soft cheeses

The contribution to flavor generation and secondary proteolysis of 2 strains of mesophilic lactobacilli isolated from cheese was studied. Miniature soft cheeses (200 g) were produced with or without the inclusion of a culture of Lactobacillus plantarum I91 or Lactobacillus casei I90 in the starter composed of Streptococcus thermophilus. During ripening, cheeses containing the added lactobacilli showed an increased content of total free amino acids, but this increase was only significant in cheeses with Lb. plantarum I91. In addition, free amino acid profiles were modified by selective increases of some amino acids, such as Asp, Ser, Arg, Leu, and Phe. Cheeses inoculated with Lb. plantarum I91 or Lb. casei I90 were also characterized by a significantly higher concentration of diacetyl, a key flavor compound, and an increased content of acetoin. Results suggest an increase in the catabolism of either citrate or aspartate, with the production of the derived aroma compounds. Overall, aspartate content increased in both lactobacilli-added cheeses, whereas citrate was more or less constant, suggesting that aspartate could be the source of increased diacetyl and acetoin. A triangle aroma test showed that the addition of the lactobacilli strains significantly changed the sensory attributes of cheeses. At least 11 of 12 panelists commented that the aroma of cheeses with adjuncts was more buttery than that of control cheeses, which is desirable in most soft cheeses. Both Lb. plantarum I91 and Lb. casei I90 performed well as adjunct cultures by influencing cheese aroma development and cheese proteolysis.     

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hard-cooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 ??m). The substrates were incubated at 34 °C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37 °C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.     

A model to assess lactic acid bacteria aminopeptidase activities in Parmigiano Reggiano cheese during ripening

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.     

Ripening of cheese made from full concentrated milk retentate with and without peptidase addition

The aim of this study was to determine ripening of cheese made from full concentrated (FC) milk retentate with and without peptidase addition. No free amino acids (FAAs) were found in FC cheese at the end of ripening. However, added peptidase increased FAA formation. Protein and peptide profile analysis showed that FAA and small peptides increased during ripening and therefore some secondary proteolysis occurred. Added peptidase increased D‐lactic acid formation during ripening of cheeses. This kind of changes in lactose fermentation should be considered during developing the making cheese with different enzyme addition.     

Lactococcal aminotransferases AraT and BcaT are key enzymes for the formation of aroma compounds from amino acids in cheese

Amino acid catabolism plays a major role in cheese aroma development. Previously, we showed that the lactococcal aminotransferases AraT and BcaT initiate the conversion of aromatic amino acids, branched-chain amino acids and methionine to aroma compounds. In this study, we evaluated the importance of these two enzymes in the formation of aroma compounds in a cheese model by using single araT and bcaT mutants and a double araT/bcaT mutant. We confirmed that addition of α-ketoglutarate, a co-substrate of aminotransferases, stimulates the conversion of amino acids to aroma compounds in cheese. The results demonstrated that AraT and BcaT are essential for conversion of aromatic and branched-chain amino acids to aroma compounds by Lactococcus lactis in the cheese model and that they also play a major role in the formation of volatile sulphur compounds from methionine. However, another pathway or another aminotransferase appears also to be weakly involved in the formation of these sulphur compounds.     

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.     

Cheddar Cheese Ripening and Flavor Characterization: A Review

Cheddar cheese is a biochemically dynamic product that undergoes significant changes during ripening. Freshly made curds of various cheese varieties have bland and largely similar flavors and aroma and, during ripening, flavoring compounds are produced that are characteristic of each variety. The biochemical changes occurring during ripening are grouped into primary events including glycolysis, lipolysis, and proteolysis followed by secondary biochemical changes such as metabolism of fatty acids and amino acids which are important for the production of secondary metabolites, including a number of compounds necessary for flavor development. A key feature of cheese manufacture is the metabolism of lactose to lactate by selected cultures of lactic acid bacteria. The rate and extent of acidification influence the initial texture of the curd by controlling the rate of demineralization. The degree of lipolysis in cheese depends on the variety of cheese and may vary from slight to extensive; however, proteolysis is the most complex of the primary events during cheese ripening, especially in Cheddar-type cheese.     

Biochemistry of cheese ripening

Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.     

Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains

Aroma development in cheese products results from the metabolic activities of cheese bacteria, by glycolysis, lipolysis and proteolysis. To respond to the increasing demand for products with improved aroma characteristics, the use of bacterial strains for cheese ripening with enhanced flavour production is seen as promising. In this review, the catabolism of amino acids, presumably the origin of some major aroma compounds, is discussed. The techniques of detection of flavour-producing strains are then presented. Their detection may be achieved either by genotyping, by enzymatic analysis, or by physico-chemical analysis such as HPLC, TLC, GC, and electronic nose.     

Cheese flavour development by enzymatic conversions of peptides and amino acids

During ripening of cheese, many biochemical processes take place, which are essential for flavour development. The breakdown of caseins is a prerequisite for flavour development. A good balance between proteolysis and peptidolysis prevents the formation of bitterness in the cheese. For this reason, it is necessary to focus on starter cultures with highly active peptidases, which should be active in the cheese matrix. Amino-acid-converting enzymes (AACEs) are involved in the degradation of amino acids, which are liberated during proteolysis. Their activity results in various volatile (flavour) components; most notably the degradation of methionine results in flavour-active sulphur compounds. AACEs involved in degradation of methionine and other amino acids were identified and their role in (cheese) flavour formation is described. At least two pathways leading to the formation of sulphur compounds were identified. Overproduction of one of the enzymes involved, results specifically in a higher formation of sulphur compounds. This result, together with the observation that flavour production is highly strain-specific amongst various lactococcal bacteria, offers a new potential for industrial applications.     

Quantification of volatile compounds in goat milk Jack cheese using static headspace gas chromatography

Goat milk Jack cheeses were manufactured with different levels of proteolytic endo- and exopeptidases from lysed bacterial cultures and aged for 30 wk. The aroma compounds that are potentially important in contributing the typical flavor of goat milk Jack cheese were quantified using static headspace gas chromatography. The concentrations of volatile compounds were evaluated every 6 wk throughout the aging period. Odor activity values of volatile compounds were calculated using the sensory threshold values reported in literature and their concentrations in Jack cheeses. Odor activity values of identified compounds were used to assess their potential contribution to the aroma of goat milk Jack cheeses. The odor activity values indicated that the ketones 2-hexanone, 2-heptanone, 2-nonanone, and 2,3-butanedione (diacetyl) were important odor-active compounds. The major odor-active acids found in this semi-hard goat milk cheese were butanoic, 2-methyl butanoic, pentanoic, hexanoic, and octanoic acids. Among the aldehydes, propanal and pentanal had high odor activity values and likely contributed to the aroma of this cheese. The concentrations of butanoic, pentanoic, hexanoic, heptanoic, octanoic, and nonanoic acids increased significantly in goat milk Jack cheese throughout aging. The extracted enzymes from lysed bacterial cultures that were added to the cheeses during manufacturing caused considerable increases in the concentrations of butanoic and hexanoic acids compared with the control. However, the lower concentration of peptidases resulted in an increased concentration of butanal, whereas more peptidases resulted in a lower concentration of 2-nonanone in goat milk Jack cheeses.     

一种植物乳杆菌对干酪模型成熟中微生物及蛋白质水解的影响

采用无菌条件下生产的新鲜干酪凝块制作干酪模型,以WSN,12%TCASN和氨基酸含量等为指标测定植物乳杆菌SP-3对其的促熟作用。微生物测定表明SP-3在干酪模型的成熟过程中(12d)能保持较高的浓度;WSN结果表明SP-3对干酪中蛋白质的初级水解无显著影响;12%TCASN的结果表明SP-3影响了干酪中小肽的形成;5%PTA-SN和总游离氨基酸浓度测定结果表明植物乳杆菌SP-3促进了体系中游离氨基酸的产生;游离氨基酸的分析显示谷氨酸、亮氨酸、组氨酸的含量明显高于对照组,而丝氨酸、赖氨酸、脯氨酸的含量低于对照组。以上结果表明,植物乳杆菌SP-3能加快干酪体系中蛋白质的水解进程,具有促进干酪成熟的作用。     

Ripening time estimation of Kariesh cheese

Kariesh cheese is a popular cheese in Egypt produced by acid coagulation of milk. It can be consumed fresh or after ripening. Proteolysis in cheese was measured by determining soluble nitrogen (SN), amino acid nitrogen (AAN), total amino acids (TAA) and free amino acids (FAA). SN, AAN and FAA increased during ripening. Free amino acids profile revealed in total 16 amino acids and the same distribution of free amino acids. Cheese ripening was influenced by the type of milk and the method of production. The mildly acid sweet flavour was attributed to the concentration of glutamic acid, aspartic acid, proline and valine. Linear regression analysis was carried out to estimate the ripening time of this cheese. A positive correlation between the accumulation of amino acid and ripening time was established. The highest coefficient of determination near one resulted from glutamic acid (R² = 0.99) followed by lysine (R² = 0.97–0.99), then aspartic acid (0.90–0.98). From the linear regression equation for glutamic acid, lysine, aspartic acid or proline, the ripening time of Kariesh cheese in weeks was determined as follows: Time of ripening [weeks] = mg amino acid 100 g cheese-a/b where b = slope and a = intercept of regression straight line at 0 time.     

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.     

Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese

Two probiotic strains, Lactobacillus acidophilus and Lactobacillus paracasei subsp. paracasei, were used as adjunct cultures in semi-hard cheesemaking experiments, in order to study their influence on proteolysis during ripening. Cheeses with and without probiotic bacteria were manufactured. The population of probiotics remained above 10⁷ cfu g⁻¹ during all ripening, and they did not influence primary proteolysis. However, L. acidophilus produced a significant increase in the level of low molecular weight nitrogen compounds and individual free amino acids; the amino acid profiles were also different. Multivariate analysis of peptide profiles showed that samples were grouped mainly by ripening time, although the impact of probiotics was also noticeable. L. acidophilus showed a clear influence on secondary proteolysis, while a minor effect of L. paracasei was evidenced at the end of the ripening. These results showed that the tested strains influenced distinctly proteolysis of cheeses, probably as a consequence of their different proteolytic systems and their activity via the alimentary matrix (cheese).     

Proteolysis and texture changes of a Spanish soft cheese (‘Torta del Casar’) manufactured with raw ewe milk and vegetable rennet during ripening

Proteolysis and textural changes of the Spanish ewe raw milk soft cheese of the Protected Designation of Origin Torta del Casar were studied in four different stages of ripening, with 1, 30, 60 and 90 days. In general, proteolysis in Torta del Casar cheese was weak at 1 and 30 days and it was more intense between the 30–60 days of ripening. Soluble nitrogen non‐protein nitrogen, polypeptide N and free amino acids values significantly increased during cheese ripening. Protein and casein nitrogen decreased significantly after 60 days of ripening resulting in the increase of the other nitrogen fractions measured. Caseins changes determined by capillary zone electrophoresis showed that proteolysis of β‐casein occurred faster than αs1‐casein but the latter suffered higher proteolytic degradation at the end of ripening (day 90). This pattern of degradation of caseins is reversed in other cheeses made with animal rennet. Texture analysis showed that firmness and consistency decreased along ripening while adhesiveness increased. Highly significant correlations were found between textural parameters, residual caseins levels and nitrogen fractions during maturation, which shows the importance of proteolytic changes for an optimal texture formation.     

Impact of chymosin- and plasmin-mediated primary proteolysis on the growth and biochemical activities of lactobacilli in miniature Cheddar-type cheeses

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of α_s1-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.