Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats

A water-soluble extract of a gouda-type cheese showed dipeptidyl-peptidase 4 (DPP-4)-inhibitory activity, which should improve glucose tolerance in type 2 diabetes. With liquid chromatography, we obtained four active fractions from the water-soluble extract and analysed the constituent peptides with liquid chromatography/mass spectrometry. Four peptides with the X-Pro-structure showed IC₅₀ values of <200 μm. β-Casein peptide residues 70-77 (β-CN f70-77; LPQNIPPL) showed the highest DPP-4-inhibitory activity, which increased during the ripening period. Glucose tolerance tests were performed in rats orally administered synthesized LPQNIPPL (30 mg 100 g⁻¹ rat weight) with a cross-over experimental design. The post-prandial area under the blood glucose curve was significantly reduced (P < 0.02) in the LPQNIPPL-administered group compared with that in the placebo-treated group. This is the first report that has identified DPP-4-inhibitory casein-derived peptides from gouda-type cheese with an effect on plasma glucose in a rat model.     

An application in Gouda cheese manufacture for a strain of Lactobacillus helveticus ND01

Gouda cheese was manufactured with Lactococcus lactis ssp. lactis IMAU60010, L. lactis ssp. cremoris IMAU40136 and L. helveticus ND01 isolated from the naturally fermented milk in China. Starter cultures added with L. helveticus ND01 produced Gouda cheese with dramatically more proteolysis than control cheeses. Compared with control cheese, experimental cheese with L. helveticus ND01 adjunct revealed dramatic increase in both Angiotensin I‐converting enzyme (ACE)‐inhibitory activity and γ‐aminobutyric acid content. The ACE‐inhibitory activity of Gouda cheeses with the addition of 1 × 10⁵ CFU/mL L. helveticus ND01 increases from 53.7 to 83.1% at 6 weeks of ripening.     

Impact of native Lactobacillus paracasei subsp. paracasei and Pediococcus spp. as adjunct cultures on sensory quality of Iranian white brined cheese

Paired wild‐type cultures consisting of a Lactobacillus paracasei subsp. paracasei (three strains) or Pediococcus pentosaceus (one strain) and a Pediococcus inopinatus (five strains) were used as adjunct cultures in the production of Iranian white brined cheese. After 8 weeks of ripening, adjunct‐treated cheeses produced by L. paracasei subsp. paracasei and P. inopinatus received significantly higher scores for flavour/taste, aroma, texture and overall preference than those produced by P. pentosaceus and P. inopinatus as well as the control cheese (P < 0.05). In conclusion, a greater improvement of sensory quality of cheeses was strongly associated with the presence of L. paracasei subsp. paracasei rather than pediococci.     

Microbial characterization of Iranian traditional Lighvan cheese over manufacturing and ripening via culturing and PCR-DGGE analysis: identification and typing of dominant lactobacilli

This paper reports on the diversity and dynamics of the dominant microbial populations during manufacturing and ripening of Lighvan, a traditional, starter-free Iranian cheese made from raw ewe and goat’s milk as determined by culturing and PCR-DGGE. Similar dominant populations, composed of Lactococcus lactis and Lactobacillus spp. strains, were found by both techniques. However, discrepancies regarding the identity of the Lactobacillus species were encountered. Lactobacillus curvatus and Lactobacillus sakei proved to be dominant by PCR-DGGE; in contrast, Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis and Lactobacillus plantarum were the majority cultivable organisms. RAPD typing of lactobacilli isolates showed wide genetic diversity among the species. Moreover, strain compositions change over time; L. brevis and L. paraplantarum were dominant in milk and were replaced by L. plantarum and L. paracasei strains as ripening progressed.     

Effect of seaweed flakes addition on the development of bioactivities in functional Camembert‐type cheese

The effect of adding Palmaria palmata or Saccharina longicruris to Camembert‐type cheese on both the antioxidant capacity (ORAC) and Angiotensin I‐converting enzyme (ACE)‐inhibitory activity has been studied with the aim of developing a functional food. The nutritional composition showed that P. palmata had the highest total protein and carbohydrate contents while S. longicruris demonstrated the highest total fibre and minerals contents. The bioactivities determined in the S. longicruris soluble extract were the highest. Three cheese model curds were studied: cheese control (CC), 2% of P. palmata (C2PP) and 2% of S. longicruris (C2SL). During ripening (20 days), the curd pH of all treatments was significantly similar, as their ORAC values, starting at 0 and reaching 41.28 mmol TE g^?1. The ACE‐inhibitory activity of the three treatments was significantly similar, at day 0 (13.20%) and day 20 (58.27%). The feasibility of including these two seaweeds into Camembert‐type cheeses was validated through the adequate development of bioactivities during ripening supporting promising food applications.     

Identification of ACE-inhibitory peptides in different Spanish cheeses by tandem mass spectrometry

Different Spanish cheeses (Cabrales, Idiazábal, Roncal, Manchego, Mahón and a goat's milk cheese), made with diverse technologies and milk from different species, were studied for their angiotensin converting enzyme (ACE)-inhibitory activity. Among the water-soluble extract WSE<1000-Da of the different samples, Cabrales cheese was the most active with an inhibitory activity of 76.1%. On the contrary, Mahón cheese showed the lowest ACE-inhibitory index (56.6%). In order to identify the peptides involved in this activity, the WSE<1000-Da were analyzed by HPLC-MS/MS and off-line MS/MS. A total of 41 major peptides were identified in the different cheeses. Most of them derived from β-and α_s1-casein. Several of these peptides were selected on the basis of the presence of proline as the C-terminal amino acid, and they were chemically synthesized. All peptides showed moderate or low ACE-inhibitory activity. Peptide DKIHP [β-CN f(47–51)], a sequence detected in all samples except Mahón cheese, showed the highest inhibitory activity with an IC₅₀ value of 113.1 μM.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Role of salt in Iranian ultrafiltered Feta cheese: Some textural and physicochemical changes during ripening

The effect of NaCl (0–4%) on the ripening of Iranian ultrafiltered (UF)‐Feta cheese was assessed over 120 days of ripening at 4 °C. Whey percentage, whey salt, whey pH, cheese pH and textural properties of hardness and cohesiveness were monitored, and experimental modelling performed using response surface methodology. Texture, pH and whey percentage were significantly affected by NaCl and ripening. The maximum whey of 22% was recorded at the end of ripening period. Texture of this cheese becomes harder during ripening confirming cheese pH and whey percentage being the major determining factors. Cheese samples were more elastic than viscous with cohesiveness values of 0.6–0.9.     

Proteolysis in model Portuguese cheeses: Effects of rennet and starter culture

To shed further light onto the mechanisms of proteolysis that prevail throughout ripening of Portuguese cheeses, model cheeses were manufactured from bovine milk, following as much as possible traditional manufacture practices – using either animal or plant rennet. The individual role upon proteolysis of two (wild) strains of lactic acid bacteria – viz. Lactococcus lactis and Lactobacillus brevis, which are normally found to high viable numbers in said cheeses, was also considered, either as single or mixed cultures. Our experimental results confirmed the influence of rennet on the proteolysis extent, but not on proteolysis depth. On the other hand, the aforementioned strains clearly improved release of medium- and small-sized peptides, and contributed as well to the free amino acid pool in cheese.     

Microbiological and biochemical changes in herby cheese during ripening

The aim of this study was to determine and compare the microbiological, biochemical and sensory characteristics of herby cheese made with two different methods. In the first method (M1), milk and herbs were pasteurized at 65°C for 30 min, and Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added as starter culture at an inoculum ratio of 1.5%. In the second method (M2), the conventional cheesemaking was applied. Microbiological and biochemical changes were monitored throughout the ripening period of 90 days. Samples were taken from cheeses on days 1, 15, 30, 60, and 90. At the end of ripening, sensory characteristics of cheeses manufactured with both methods were evaluated. The obtained results suggested that most changes in pH, titratable acidity, and dry matter contents of cheese varieties were not found to differ statistically significant, but the difference in salt content was significant (P < 0.01). Total aerobic count, lactic acid bacteria, Staphylococcus aureus, coliforms, moulds, yeasts, proteolytic and lipolytic microorganism counts were lower in M1 cheese samples than those of M2 cheese samples (P < 0.01). The numbers of psychrotrophic microorganism in both cheese types were not found to differ significantly. Moreover, the results suggested that there were significant differences (P < 0.01) in the degrees of proteolysis and lipolysis of the cheese varieties. High proteolysis and lipolysis rates were monitored in the traditional cheese samples. However, there were no significant differences between the sensory characteristics of cheese samples.     

Effects of Mentha longifolia L. essential oil on viability and cellular ultrastructure of Lactobacillus casei during ripening of probiotic Feta cheese

The effects of Mentha longifolia L. essential oil during ripening and storage probiotic Feta cheese were studied, in relation to viability and cellular ultrastructure of Lactobacillus casei. The addition of the essential oil in the concentrations from 0.0 to 0.03% was trialled: the 0.03% treatment resulted in the highest viability of L. casei and the lowest pH value compared with other treatments (P < 0.05). Electron microscopy showed that essential oil caused no harm to L. casei. This study demonstrated the favourable effects of M. longifolia on optimal maintenance of L. casei at the end of cheese storage period.     

Proteolysis in ultra-filtered and conventional Iranian white cheese during ripening

Conventional and ultra-filtered (UF) Iranian white cheeses were made with almost identical gross chemical composition and the extent and characteristics of proteolysis were studied during ripening. UF cheeses exhibited a lower rate of development of pH 4.6-soluble nitrogen than conventional cheeses. The rates of degradation of α_s1-casein and particularly β-casein were lower in UF cheeses than in conventional cheeses. Plasmin activity was lower in UF cheeses than that in conventional cheese, whereas coagulant activity was higher in the former. Noticeable qualitative and quantitative differences were observed in reverse-phase high performance liquid chromatography (RP-HPLC) peptide profiles between UF and conventional white cheeses and chemometric analysis of peak height data distributed the cheeses into two separate groups. The levels of free amino acids in UF cheeses were lower than in conventional cheeses. Lower peptide degradation and production of amino acids suggested slower ripening, which may have been associated with the weak aroma development characteristic of UF cheeses.     

Effect of a nisin-producing lactococcal starter on the late blowing defect of cheese caused by Clostridium tyrobutyricum

Clostridium tyrobutyricum causes swelling, cracks and off-flavours of cheeses (late blowing defect, LBD) due to butyric acid fermentation. To control this spoilage bacterium, we investigated the use of nisinogenic Lactococcus lactis subsp. lactis INIA 415 as starter in cheeses contaminated with C. tyrobutyricum spores. Control cheese made with spores showed LBD after 14 days of ripening. However, in cheese made with the bacteriocin producer and spores, in which (unlike control cheese) bacteriocin activity was detected throughout ripening, LBD occurred after 21 days. At this stage, level of lactic acid was 1.22-fold higher (P < 0.01) and concentrations of propionic and butyric acids were 2.15- and 2.32-fold, respectively, lower (P < 0.01) in cheese made with the nisin producer than in control cheese, according to the less pronounced spoilage symptoms showed by the former cheese. The bacteriocin producer delayed the appearance of LBD, although it cannot arrest completely C. tyrobutyricum growth.     

Survival of micro‐organisms and organic acid profile of probiotic Cheddar cheese from buffalo milk during accelerated ripening

The study aimed to assess the impact of ripening at elevated temperatures on the survival of probiotic micro‐organisms and production of organic acids in Cheddar cheese. Cheese was manufactured from buffalo milk using lactococci starters along with different probiotic bacteria (Lactobacillus acidophilus LA‐5, Bifidobacterium bifidum Bb‐11 and Bifidobacterium longum BB536) as adjunct cultures. The cheeses were ripened at 4–6 °C or 12–14 °C for 180 days and examined for composition, organic acids and microbial survival. The production of organic acids was accelerated at 12–14 °C when compared to normal ripening temperatures. The probiotic bacteria increased production of lactic and acetic acids, compared to cheese made with lactococci alone. The survival of the mesophilic starters was significantly (P < 0.05) reduced in all the cheese samples ripened at the higher temperature. However, the probiotic bacteria remained viable (>7.0 log₁₀ cfu/g) throughout the 180 days of ripening, irrespective of temperature. It was concluded that Cheddar containing additional probiotic cultures can effectively be ripened at elevated temperatures without any adverse effects.     

Lysis of Lactobacillus casei 5Mn 373 accelerates Grana cheese ripening

The influence of the adjunct of a peptidolytic Lactobacillus casei strain on Grana cheese ripening was studied. Strain erythromycin resistance enabled the monitoring of its growth and death kinetics during cheese maturation. Cell lysis was estimated by the dosage of intracellular X-prolyl-dipeptidyl aminopetidase in cheese extracts. L. casei growth reached a maximum level after the second month of cheese ripening when the initial added cell level was 5×10⁵ cfu/g cheese, while L. casei counts decreased from the beginning of the ripening period when the initial added cell level was 4.5×10⁷ cfu/g cheese. The maximum death rate occurred two months after the maximal cell growth, and bacterial lysis was observed approximately two months later. The characteristic amino acid pattern of control Grana cheese was obtained for all of the mature experimental cheeses independently of the inoculum size, and more rapidly when higher amounts of inocula were used due to L. casei cell lysis. The adjunct of the L. casei strain to cheese-milk substantially shortened the ripening time with no negative effects on cheese-making, chemical gross composition or flavour in the mature experimental cheeses compared to the control.     

Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses

The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilusLAFTI^® L10 during ripening at 4 and 8 °C for 24 weeks. ACE-inhibitory activity of the cheeses was maximum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI^® L26 or Lb. acidophilus LAFTI^® L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8 °C. The IC₅₀ of cheeses ripened at 4 °C was not significantly different (P > 0.05) to that ripened at 8 °C. The lowest value of the IC₅₀ (0.13 mg mL⁻¹) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI^® L10. Several ACE-inhibitory peptides were identified as κ-CN (f 96–102), α_s1-CN (f 1–9), α_s1-CN (f 1–7), α_s1-CN (f 1–6), α_s1-CN (f 24–32) and β-CN (f 193–209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides.     

Physicochemical differences between Croatian cheese matured in a lamb skin sack (Sir iz misine) and cheese matured in a rind throughout ripening

The aim of this study was to research differences in physicochemical parameters between Croatian cheese in a lamb skin sack (Sir iz misine) and cheese in a rind throughout ripening. Cheese in a sack had significantly (P < 0.05) lower content of total solids, fat, proteins and salt which showed the ‘protective’ effect of skin sack and higher permeability of natural rind. The water‐soluble nitrogen in the total nitrogen (%TN) and 12% trichloroacetic acid‐soluble nitrogen (%TN) at the end of ripening was significantly (P < 0.05) higher in cheese in a sack than in cheese in a rind which indicates intensive proteolysis in cheese in a sack.     

A preliminary study on the role of alkaline phosphatase in cheese ripening

A preparation of exogenous alkaline phosphatase (ALP), containing 17,500 mU L⁻¹, was added to pasteurized milk (PM) to study its role in cheese ripening. Three miniature Cheddar-type cheeses were made from PM containing no added ALP (control), PM plus 23 μL ALP (T1), to give ALP concentration similar to that in raw milk, and PM plus 46 μL ALP (T2). Milk, after addition of ALP, was held at 6 °C for 12 h before cheese manufacture and the experiment was replicated three times. The control, T1 and T2 milks contained ALP activity of 415, 2391 and 4705 mU L⁻¹, respectively. The addition of ALP to PM caused significant (P<0.05) changes in moisture content of miniature cheeses but did not cause any changes in protein content. Levels of water-soluble N during ripening of the cheeses were similar for control, T1 and T2 cheeses. The concentration of amino acids was not affected by the level of ALP present in milk. However, reversed-phase HPLC showed differences in the peptide patterns of control, T1 and T2 cheeses, suggesting a role of ALP in cheese ripening. The results suggest that ALP may play a role in cheese ripening, but further studies are needed to confirm this.     

Proteolysis in goat cheese made from raw,pasteurized or pressure-treated milk

Primary and secondary proteolysis of goat cheese made from raw (RA), pasteurized (PA; 72 °C, 15 s) and pressure-treated milk (PR; 500 MPa, 15 min, 20 °C) were examined by capillary electrophoresis, nitrogen fractionation and HPLC peptide profiles. PA milk cheese showed a more important hydrolysis (P<0.05) of α_s1-casein than RA milk cheese at the first stages of ripening (15 days), while PR milk cheese had a level between those seen in PA and RA milk cheeses. Degradation of β-casein was more important (P<0.05) in PA and PR than in RA milk cheeses at 15 days of ripening. However, from thereon β-casein in PR and RA milk cheeses was hydrolyzed at essentially similar rates, but at lower rates (P<0.05) than in PA milk cheeses. Pressure treatment could induce proteolysis of β-casein in a way, which is different from that produced by heat treatment. There was an increase in 4.6-soluble nitrogen (WSN) and in trichloroacetic acid (TCASN) throughout ripening in cheeses, but higher contents (P<0.05) in PA and PR milk cheeses at the end of ripening were observed. PR milk cheeses contained considerably higher content (P<0.05) of free amino acids than RA or PA milk cheeses. In general, heat and pressure treatments had no significant effect on the levels of hydrophobic and hydrophilic peptides.     

Ultrafiltered milk reduces bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide-producing culture

The objectives were to reduce bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide (EPS)-producing culture and study relationships among ultra-filtration (UF), residual chymosin activity (RCA), and cheese bitterness. In previous studies, EPS-producing cultures improved the textural, melting, and viscoelastic properties of reduced-fat Cheddar cheese. However, the EPS-positive cheese developed bitterness after 2 to 3 mo of ripening due to increased RCA. We hypothesized that the reduced amount of chymosin needed to coagulate UF milk might result in reduced RCA and bitterness in cheese. Reduced-fat Cheddar cheeses were manufactured with EPS-producing and nonproducing cultures using skim milk or UF milk (1.2×) adjusted to a casein:fat ratio of 1.35. The EPS-producing culture increased moisture and RCA in reduced-fat Cheddar cheese. Lower RCA was found in cheese made from UF milk compared with that in cheese made from control milk. Ultrafiltration at a low concentration rate (1.2×) produced EPS-positive, reduced-fat cheese with similar RCA to that in the EPS-negative cheese. Slower proteolysis was observed in UF cheeses compared with non-UF cheeses. Panelists reported that UF EPS-positive cheese was less bitter than EPS-positive cheese made from control milk. This study showed that UF at a low concentration factor (1.2×) could successfully reduce bitterness in cheese containing a high moisture level. Because this technology reduced the RCA level (per g of protein) to a level similar to that in the control cheeses, the contribution of chymosin to cheese proteolysis would be similar in both cheeses.