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.     

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.     

Pre-treatment methods of Edam cheese milk. Effect on cheese yield and quality

The effect of high-temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) on the Edam vat milk composition, processing and cheese yield, ripening and functional characteristics were studied. The protein level of the MF and UF cheese milk was adjusted to 42 g/kg, whereas the level in the reference (REF) and HH milk was 34 g/kg. The cheese yield from ultrafiltration and microfiltration milk (CY_v) was 12.8 g/100 g milk, yield from reference and high-temperature heat treatment milk was 10.1 and 10.2 g/100 g milk, respectively. The adjusted cheese yield (ACY_r), calculated from raw milk, was lowest when MF was used. The pre-concentration method had little effect on the starter activity: no differences were observed in the pH of cheeses. The compositions of the ripened cheeses were comparable. The casein to fat ratio of MF cheese was elevated, possibly due to elevated casein to fat ratio of vat milk. Even though the high-temperature heat treatment, ultrafiltration and microfiltration cheeses were harder than reference cheese, they retained their elasticity. Resilience was significantly higher with microfiltration and ultrafiltration cheeses. The sensory quality of all cheeses was considered according to specification. The pre-treatment methods had little effect on the processing characteristics, cheese quality or yield when calculated on the basis of the quantity of original milk.     

Angiotensin-converting enzyme inhibitory activity of water-soluble extracts of Asiago d'allevo cheese

The angiotensin-converting enzyme (ACE) inhibitory activity of water-soluble extracts (WSE) from Asiago cheeses was assayed in two cheese production systems and with different ripening times. The WSE were ultrafiltered through 10 kDa and 3 kDa cut-off membranes to evaluate the ACE inhibitory activity of long and short peptides, respectively. The cheese production systems had no significant effect on the ACE inhibitory activity, whereas 6-month-old cheeses had higher inhibitory potency than the more ripened ones. Moreover, the fraction containing peptides smaller than 3 kDa made a more considerable contribution to ACE inhibitory activity than the fraction smaller than 10 kDa, suggesting an inhibitory effect due to short peptides. The peptidic fraction was characterized using RP-HPLC coupled to mass spectrometric detection. Simulated gastrointestinal digestion was carried out to evaluate the effects of digestive enzymes on the generation of bioactive peptides, but this did not significantly affect the inhibitory activity.     

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D–spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC–tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D–spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.     

Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates

Several research studies have shown that protein hydrolysates from milk and soy contain peptides that possess angiotensin I converting enzyme (ACE) inhibitory properties and may help to prevent hypertension. To date, no studies have been conducted to determine if red lentil (Lens culinaris) proteins contain peptides with ACE-inhibitory properties. The objective of the present work was to characterize the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACE-inhibitory properties. Red lentil protein extracts were prepared and fractionated to obtain enriched albumin, legumin and vicilin fractions. Protein/peptide profiles were studied by electrophoresis and ACE-inhibitory activity was measured using the HPLC hippuryl-His-Leu (HHL) substrate method. Our results revealed that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the ACE-inhibitory property of the hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest half maximal inhibitory concentration (IC₅₀) (111 ± 1 μmol/L) (i.e., highest ACE-inhibitory activity), followed by the enriched legumin (119 ± 0.5 μmol/L), albumin (127 ± 2 μmol/L) and vicilin (135 ± 2 μmol/L) fractions, respectively.     

Proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet

Powdered plant coagulant (PPC) obtained from the cardoon (Cynara cardunculus) was compared with calf rennet (CR) for the manufacture of goats’ milk cheese, by determining difference in the proteolysis throughout ripening. There were no substantial differences between the compositions of cheeses made using the two types of coagulants. However, cheeses manufactured with PPC exhibited higher levels of pH 4.6-SN than cheese made using CR. The extent of breakdown of α_s-casein, as measured by urea-PAGE, was greater in cheese made using PPC than cheese made using CR. The formation of hydrophobic peptides and the ratio of hydrophobic/hydrophilic peptides throughout the ripening were higher in cheeses made with PPC than in cheeses made with CR. Principal component analysis (PCA) of peak heights of RP-HPLC peptide profiles of the ethanol-soluble and ethanol-insoluble fractions distributed the samples according to the coagulant used in their manufacture. Quantitative differences in several peptides were evident between the two types of cheese.     

Use of changes in triacylglycerols during ripening of cheeses with high lipolysis levels for detection of milk fat authenticity

The triacylglycerol (TAG) compositions by carbon number during ripening of two Protected Designation of Origin (PDO) cheeses were analysed using short capillary column gas chromatography. Lipolysis levels were high in the Cabrales (blue cheese produced from cows’ milk or from blends of cows’ with goats’ milk) and Majorero goats’ milk cheeses at the end of ripening, with free fatty acid (FFA) levels of around 24 000 ppm and significant changes in the TAG composition. The level of lipolysis in an industrial blue cheese made from ewes’ milk was low, with an FFA value of around 6000 ppm and no significant changes in the TAG composition during ripening. The TAG values recorded for each cheese sample were substituted into the multiple regression equations that have been proposed for use in detecting foreign fats in milk fat. The values thus obtained were within the established ranges in early ripening. In the cheeses with high lipolysis levels during ripening, some of the values obtained fell outside the established ranges. These equations can be potentially useful for detecting foreign fats in these cheeses, when employed early in the ripening period. Furthermore, it is important to take into account that before coming to a conclusion about cheese authenticity, several individual samples should be analysed.     

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Study of the chemical composition,proteolysis, volatile compounds,and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese

The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and α_s-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for α_s-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.     

Proteolysis, lipolysis, volatile compounds, texture, and flavor of Hispánico cheese made using frozen ewe milk curds pressed for different times

Hispánico cheese is manufactured in Spain from a mixture of cow and ewe milk. Production of ewe milk varies throughout the year, with a peak in spring and a valley in summer and autumn. To overcome this seasonal shortage, curd from spring ewe milk may be frozen and used for cheese manufacture some months later. In the present work, ewe milk curds pressed for 15, 60, or 120 min were held at −24°C for 4 mo, thawed, cut to 1-mm pieces, and mixed with fresh cow milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same (80:20) proportion. Cheeses, made in duplicate experiments, were analyzed throughout a 60-d ripening period. No significant differences between cheeses were found for lactic acid bacteria counts, dry matter content, hydrophilic peptides, 47 out of 68 vol.tile compounds, texture, and flavor characteristics. On the other hand, differences of minor practical significance between experimental and control cheeses, unrelated to the use of frozen ewe milk curd or the pressing time of ewe milk curd, were found for pH value, aminopeptidase activity, proteolysis, hydrophobic peptides, free amino acids, free fatty acids, and the remaining 21 vol.tile compounds. It may be concluded that the use of frozen ewe milk curd in the manufacture of Hispánico cheese does not alter its main characteristics.     

Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified refrigerated milk

 The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO₂-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO₂-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO₂-acidified and non-acidified refrigerated or fresh milk, indicating that CO₂ treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO₂-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO₂-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO₂ can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses. Received: 1 July 1997 / Revised version: 1 September 1997     

Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified refrigerated milk

 The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO₂-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO₂-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO₂-acidified and non-acidified refrigerated or fresh milk, indicating that CO₂ treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO₂-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO₂-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO₂ can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses. Received: 1 July 1997 / Revised version: 1 September 1997     

Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores

This paper reports the technological effects of inoculating Cabrales cheese (a traditional, Spanish, blue-veined cheese) with Penicillium roqueforti spores. Three batches of inoculated Cabrales cheese were manufactured and a number of their microbial and biochemical variables recorded. The results were compared with those obtained for three batches of control cheese made using traditional technology (i.e., adding neither starter cultures nor fungal spores). Although mould and yeast populations grew more quickly in the inoculated cheeses, their normally dominant and representative microbial populations were not affected neither was their gross biochemical composition changed. The variations observed were thought to be caused by the uncontrolled environmental conditions of manufacture and ripening. The development of free amino acids and volatile compounds was significantly increased at 30 days in the inoculated cheeses, although the values for both types of cheese were almost identical at 90 days. The inoculated cheeses obtained higher scores in a hedonistic sensorial evaluation. Thus, inoculation improved the standardization and quality of the cheese.     

PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese

The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem.     

Hot topic: Changes in angiotensin-converting enzyme inhibition and concentrations of the tripeptides Val-Pro-Pro and Ile-Pro-Pro during ripening of different Swiss cheese varieties

The angiotensin-converting enzyme (ACE) inhibitory activity and the concentration of the 2 ACE-inhibiting tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) were studied during cheese ripening in 7 Swiss cheese varieties. The semi-hard cheeses Tilsiter, Appenzeller 1/4 fat, Tête de Moine, and Vacherin fribourgeois and the extra-hard and hard cheeses Berner Hobelkäse, Le Gruyère, and Emmentaler were investigated. Three loaves of each variety manufactured in different cheese factories were purchased at the beginning of commercial ripeness and investigated at constant intervals until the end of the usual sale period. Good agreement was found between ACE-inhibitory activity and the total concentration of VPP and IPP at advanced ripening stages. In most of the investigated varieties ACE-inhibitory activity and the concentration of the 2 tripeptides initially increased during the study period. A decline in the concentration of VPP and IPP was obtained toward the end of the investigated period for Tilsiter and Gruyère. The ratio of VPP/IPP decreased during ripening in all varieties with the exception of Emmentaler. However, large variations were observed among the cheese varieties as well as the individual loaves of the same variety. Chemical characterization of the investigated cheeses revealed that qualitative differences in the proteolysis pattern, not quantitative differences in the degree of proteolysis, are responsible for the observed variations in the concentrations of VPP and IPP. The presence of Lactobacillus helveticus in the starter culture was associated with elevated concentrations of VPP and IPP. The results of the present study show that concentrations of VPP and IPP above 100 mg/kg are attainable in semi-hard cheese varieties after ripening periods of about 4 to 7 mo and that stable concentrations of the 2 antihypertensive tripeptides can be expected over several weeks of cheese ripening.     

The Effect of Yeast Species from Raw Milk in China on Proteolysis and Aroma Compound Formation in Camembert-Type Cheese

The influence of selected yeast strains (Pichia fermentans, Issatchenkia orientalis, Yarrowia lipolytica) on Camembert-type cheeses has been investigated by monitoring their microbial and compositional changes. During the ripening period from day 1 to day 15, the yeast strains grew exponentially and then slowed to a moderate growth rate. Our results indicated that the selected strains did not significantly affect the general characteristics of cheese but rather the content and ratio of individual free amino acids (P < 0.05). In the cheese with added I. orientalis, a greater amount of small peptides and a higher concentration of non-protein nitrogen and NH₃ were found. Meanwhile, the presence of adjunct yeasts did not enhance the lipolysis activity in cheese. A principal component analysis of 30 free fatty acids and volatile compounds differentiated five cheeses out of eight according to the content of specific free fatty acids and volatile compounds. In terms of the ripening period, two groups of cheeses at 15 and 45 days were distinguished.     

Angiotensin-converting enzyme inhibitory peptides from goats' milk released by in vitro gastro-intestinal digestion

The aim of this study was to identify the angiotensin-converting enzyme (ACE) inhibitory peptides released after in vitro gastro-intestinal digestion of skimmed goats' milk. The experimental approach involved a recently developed harmonised static in vitro digestion model, with mass spectrometry (MS) to identify bioactive peptides. Peptides in the post-pancreatic digest were extracted by ultrafiltration and isolated by reversed-phase high-performance liquid chromatography followed by MS. Among the identified sequences, eighteen were identical to known bioactive peptides with ACE-inhibitory activity. Peptides with dipeptidyl peptidase IV-inhibitory and antioxidant activities were also identified. The antihypertensive tripeptides valine-proline-proline and isoleucine-proline-proline were released from goats’ milk protein during in vitro gastro-intestinal digestion at concentrations of 1829.8 ± 216.4 and 141.4 ± 15.1 μg L⁻¹, respectively. This research underlines the suitability of the harmonised digestive model system to study the release of short bioactive peptides during gastro-intestinal transit.