Effects of heat treatment, starter culture, plant coagulant and ripening on in vitro ACE inhibitory and antioxidant activity of goat cheese

Effects of heat treatment, starter culture, plant coagulant and ripening on in vitro ACE inhibitory and antioxidant activity of goat cheese | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of heat treatment, starter culture, plant coagulant and ripening on in vitro ACE inhibitory and antioxidant activity of goat cheese HASAN UZKUÇ, YONCA KARAGÜL YÜCEER This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6542067/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 23 You are reading this latest preprint version Abstract The objective of this study was to determine effects of heat treatment, starter culture addition, coagulant origin and ripening time on antioxidant and angiotensin-converting enzyme-inhibitory (ACE-I) activities of goat cheese. The bioaccessibility of the cheeses was tested in the low molecular weight (< 3 kDa) fractions (LP) of in vitro digested samples containing bioactive peptides. The antioxidant activity of the cheeses was tested with ABTS [2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)] radical scavenging and CUPRAC (Cupric reducing antioxidant capacity) assays. The interaction of heat treatment and starter culture addition affected the bioaccessibility of the cheeses just as ripening process, while different coagulants had no effect. The highest ACE-I activities observed in heat-treated + starter-added milk cheeses on the 1st day and starter-free raw milk cheeses on 30th, 60th, 90th days of ripening. Starter-free raw milk cheeses exhibited better antioxidant activities in the ABTS assay on the 1st and 30th days, and CUPRAC assay throughout all ripening days. Observations indicated that raw milk cheeses fermented by natural microbiota may exhibit an antihypertensive effect with over 50% ACE-I activity and reduce oxidative damage thanks to their bioactive peptides, demonstrating potential positive impacts on human health and suggesting their possible use in functional diets. Bioactive peptide bioaccessibility bioavailability bioactivity antihypertensive Figures Figure 1 Introduction There are 10.3 million goats in Turkey, which has 1% of the world's goat population, and 2.5% of the total milk production (21.5 million tons) is goat milk [ 1 ]. World goat milk production, which was 10 million tons in 2010, doubled in 2020 and reached around 20.6 million tons [ 2 ]. This increase in the amount of goat milk production can be attributed to the increasing world population, the increasing awareness of consumers and interest in functional foods, and the rapid increase in the demand for goat milk. In recent years, researchers have reported that consumers have shown interest in goat milk and its products due to their high nutritional values ​​and the presence of bioactive components important for a healthy life [ 3 , 4 ]. Goat milk stands out with its casein micelles have smaller size, higher solubility, lower sedimentation rate, lower heat stability, and higher calcium and phosphorus content than cow milk [ 5 , 6 ], and also the fact that goat milk contains lower concentrations of α s1 -casein and higher concentrations of K-casein and β-casein contributes to its easy digestibility and lower allergenicity [ 7 , 8 ]. Goat milk proteins, due to the extensive genetic polymorphism of goats, might have different amino acid composition, which can lead to significant differences in the sensory, digestibility and bioactive properties of goat milk products [ 7 , 9 ]. Goat milk is used in various products such as fermented food, functional foods, and cosmetics. Certainly, one of the most effective utilization and ripening methods of goat milk is cheese making [ 10 ]. Cheeses are not only known for their rich content of high- quality proteins, lipids, vitamins, and essential minerals, but they also harbor bioactive components, such as bioactive peptides [ 11 ]. These peptides support various body functions, but they are initially inactive within the protein structure and are released or activated through proteolysis processes [ 11 , 12 ]. Hydrolysis processes of the goat milk proteins have been reported to produce peptides with antihypertensive, antioxidant, hypoglycemic, anti-inflammatory, antimicrobial, opioid or immunomodulatory activities [ 13 , 14 ]. In cheeses, bioactive peptides are released by the action of the milk coagulation enzyme, as well as by the activity of starter and non-starter lactic acid bacteria and act as potential antioxidants and ACE inhibitors[ 15 ]. Bioactive peptides which have the antioxidant properties reduce oxidative damage by reacting with free radicals or inhibiting oxidative reactions. Free radicals are highly unstable molecules that can easily interact with other compounds and cause oxidative damage in the body, which has been linked to various diseases such as arthritis, diabetes, and cancer [ 16 ]. Otherwise, antihypertensive activity of peptides is given through the inhibition of ACE, preventing the conversion of angiotensin I to angiotensin II, which induces the release of aldosterone, increasing sodium concentration and blood pressure; and inactivating the vasodilator bradykinin, protecting the body against cardiovascular disease [ 17 ]. ACE-I and antioxidant activity have been reported to be strongly associated with ongoing proteolysis, bioactive and non-bioactive peptides during cheese production and ripening [ 16 , 18 , 19 ]. It has been stated that the milk source used in production, the technological processes applied in production, and the strain selection of bacteria involved in proteolysis were effective on the enzyme activation of bioactive peptides in cheese [ 20 ]. Considering the diversity in production parameters, many bioactive peptides originating from milk proteins can be obtained through cheese production, and by revealing the effects of cheese production parameters, the production of bioavailable cheeses can be optimized without using additional additives. On the other hand, the functional properties of peptides released during the cheese production process depend on the changes the peptides undergo during gastrointestinal digestion and subsequent absorption [ 21 ]. Since the presence of bioactive peptides in foods does not guarantee the physiological effect of these peptides after digestion, in vivo and in vitro studies are required to prove the potential health benefits resulting from cheese consumption. Since the last 20 years, very few studies have focused on the investigation of goat milk proteins and their fractions, especially for the presence of bioactive peptides. For this reason, it is thought that there is still undiscovered information in goat milk and its products, mainly in the field of bioactive peptides [ 22 ]. Although a large number of peptides of milk protein origin have been identified, it has been reported that very few of these peptides show proven antihypertensive properties under in vitro conditions [ 23 , 24 ]. Goat milk and kefir displayed about 7-fold antioxidant activity after digestion [ 25 ]. Therefore, it is important to reveal the bioaccessibility of peptides by conducting research on the antioxidant and ACE-I activities of goat cheeses under in vitro gastrointestinal digestion conditions. In recent decade a few studies undertaken in vitro ACE-I and antioxidant activity of goat cheeses [ 26 – 31 ]. However, some studies investigated ACE-I and antioxidant potential of goat cheese in low (< 3 kDa) molecule weight fractions [ 27 , 32 , 33 ]. This study aimed to contribute to the literature by introducing the combined effects of heat treatment, starter culture addition, plant coagulant and ripening processes on the in vitro ACE-I and antioxidant activities of semi-hard goat cheese < 3 kDa peptide fractions. Materials and methods Cheesemaking Raw and 90 ºC 10 min heat-treated Turkish Saanen goat’s milk (İmbroz Mutlu Keçiler Çiftliği, Gökçeada, Türkiye) was used to produce cheeses without adding starter culture and with adding a mesophilic-thermophilic starter culture (CHOOZIT MA 4002, Danisco, Denmark). Goat milk was coagulated with a plant sourced rennet originated from Cynara cardunculus L. (Coagulante Vegetal, Pontevedra, Spain) or a calf rennet (Animal Rennet, Rumeli Maya, İstanbul Türkiye). Cheese ripening process caried out at 6–8 ºC for 90 days. Cheeses were produced from a single batch of goat milk in duplicate trials, as detailed in our previous study [ 10 ], and the cheesemaking parameters are presented in Table 1 . The cheeses were analyzed for bioaccessibility properties at 1st, 30th, 60th and 90th days of ripening. Cheeses were coded by using abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet. Table 1 Technological parameters of goat cheeses Material & Process Parameters Cheese Milk Turkish Saanen goat milk (120 L), 6.49 pH Milk composition 12.1% total solids, 3.5% protein, 3.5% fat Heat treatment No heat (R) or 90°C 10 min (H) Cooling temprature 34°C Starter culture Without starter (RF, HF)* or Lactococcus lactis subsp. lactis + Lactococcus lactis subsp. cremoris + Lactococcus lactis subsp. lactis biovar. diacetylactis + Streptococcus thermophilus (RS, HS)* CaCl 2 0.04% Maturation of milk 30–45 min Coagulant Cynara cardunculus L . protease (100 IMCU g − 1 ) (RF1, RS1, HF1, HS1)* or Calf rennet (180 IMCU mL − 1 ) (RF2, RS2, HF2, HS2)* Clotting time 90 min Cutting of the curd 3 cm 3 Resting 10 min Wheying-off 20 min Pressing 3100 Pa for 30 min + 6200 Pa for 60 min Saalting into brine 12% NaCl-12-14 h at 27.5°C Cutting of the cheese 4 equal parts Packing Plastic vaccum packing material Ripening 6–8°C – 90 days *R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet , codes indicate process factor in cheesemaking. Cheese analyses Cheese samples were analyzed for total solids (TS), protein (expressed as protein in TS (protein/TS)) and salt (expressed as salt in moisture (Salt/M)) according to [ 34 ] and fat (expressed as fat in TS (fat/TS)) according to [ 35 ] at 1st, 30th, 60th and 90th days of ripening. Chemical composition of fresh cheeses (day 1) was adapted from Uzkuç and Karagül Yüceer [ 10 ] and marked (*) in Table 2 . Table 2 Chemical composition of goat cheeses Parameter Day Cheese RF1 RF2 RS1 RS2 HF1 HF2 HS1 HS2 TS 1* 45.81 ± 0.90 Ba 46.48 ± 0.46 Ba 47.22 ± 1.02 Ba 48.12 ± 0.32 Ba 40.06 ± 0.44 Bb 38.56 ± 0.68 Bb 47.94 ± 0.72 a 47.78 ± 1.22 a 30 46.86 ± 0.85 ABa 47.17 ± 0.23 Ba 47.07 ± 0.64 Ba 47.33 ± 0.21 Ba 40.89 ± 0.17 Bb 41.80 ± 0.32 ABb 47.28 ± 0.51 a 47.33 ± 0.46 a 60 47.07 ± 0.56 ABa 46.84 ± 0.31 Ba 47.34 ± 0.23 Ba 47.33 ± 0.36 Ba 42.76 ± 0.50 Ab 43.51 ± 1.56 Ab 46.13 ± 0.39 a 47.88 ± 0.66 a 90 50.15 ± 0.49 Aab 48.97 ± 0.50 Aab 50.66 ± 0.54 Aa 51.69 ± 0.78 Aa 43.67 ± 0.18 Ad 44.45 ± 0.62 Ad 47.86 ± 0.44 b 48.17 ± 0.64 b Fat/TS 1* 48.62 ± 1.20 51.92 ± 0.57 51.96 ± 1.27 51.69 ± 0.35 48.05 ± 0.48 B 47.70 ± 1.00 B 50.10 ± 0.75 51.56 ± 0.68 30 50.13 ± 0.60 51.41 ± 0.50 52.27 ± 1.09 52.23 ± 1.69 50.46 ± 2.49 B 49.37 ± 1.71 B 51.02 ± 1.57 52.26 ± 1.54 60 49.72 ± 1.38 50.99 ± 0.88 54.12 ± 0.69 54.41 ± 1.02 50.01 ± 1.65 B 50.51 ± 1.01 AB 53.10 ± 1.03 52.71 ± 1.12 90 54.88 ± 2.11 54.90 ± 1.31 54.59 ± 1.11 54.44 ± 1.42 56.66 ± 0.79 A 56.89 ± 2.32 A 55.90 ± 1.26 54.73 ± 1.07 Protein/TS 1* 47.28 ± 1.65 A 45.39 ± 1.08 A 45.14 ± 1.90 A 48.41 ± 1.11 A 47.95 ± 1.58 A 49.33 ± 1.09 A 49.00 ± 1.13 A 48.61 ± 0.38 A 30 45.59 ± 0.84 AB 45.80 ± 0.73 A 46.25 ± 0.65 A 45.19 ± 1.21 AB 42.58 ± 1.34 AB 43.20 ± 1.97 B 45.76 ± 2.32 AB 45.07 ± 1.51 AB 60 45.47 ± 1.70 ABa 43.90 ± 0.66 ABa 42.25 ± 0.34 ABab 43.09 ± 1.37 BCab 39.53 ± 1.11 Bb 39.89 ± 0.93 Bb 45.69 ± 2.86 ABa 41.84 ± 0.78 Ba 90 40.92 ± 0.71 Bab 41.52 ± 0.89 Bab 40.32 ± 0.23 Bab 38.93 ± 0.99 Cab 38.20 ± 1.34 Bb 38.92 ± 1.23 Bb 42.42 ± 1.56 Ba 41.60 ± 1.04 Ba Salt/M 1* 5.53 ± 0.31 ab 5.84 ± 0.34 ab 5.94 ± 0.10 ab 5.86 ± 0.25 ab 6.54 ± 0.04 a 5.77 ± 0.35 a 5.13 ± 0.32 b 5.40 ± 0.09 b 30 5.93 ± 0.14 5.78 ± 0.07 5.23 ± 0.56 5.31 ± 0.17 6.24 ± 0.20 6.08 ± 0.15 5.48 ± 0.16 4.89 ± 0.27 60 6.00 ± 0.08 6.32 ± 0.46 4.96 ± 0.15 4.89 ± 0.22 5.91 ± 0.27 5.99 ± 0.16 4.76 ± 0.09 4.82 ± 0.09 90 5.88 ± 0.10 5.85 ± 0.21 4.93 ± 0.40 5.12 ± 0.35 5.77 ± 0.32 5.92 ± 0.86 5.04 ± 0.31 5.29 ± 0.09 Values are expressed as mean ± standard error. *Data (day 1) was adapted from Uzkuç & Karagül Yüceer, (2023). Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking; TS, % total solids; Fat/TS, fat in total solids; Protein/TS, protein in total solids; Salt/M, salt in moisture. Statistically significant (P ≤ 0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter. Simulated in vitro digestion In vitro digestion of cheese samples was carried out according to a standardised static in vitro digestion method suitable for food proposed by Minekus et al. [ 36 ], using enzymes α-amylase from porcine pancreas (A1031), pepsin from porcine gastric mucosa (P7012), pancreatin from porcine pancreas 8xUSP (P7545) and porcine bile extract (B8631). After in vitro digestion procedure, a centrifuge process was carried out at 9000 rpm and middle layers of centrifuged samples was used for determination of degree of hydrolysis and low molecular weight (< 3 kDa) fractions (LP). LP containing potential bioactive peptides in the samples were fractionated using Vivaspin Turbo 15 (Sartorius, Gloucestershire, United Kingdom) centrifugal filter units with a cut-off of 3 kDa (3000 MWCO) in accordance with the filter manufacturer's instructions. Assessment of hydrolysis degree (DH) The amino acid and peptide concentration (serine equivalent) of in vitro digested samples was used as an indication of protein hydrolysis in the end the pancreatic phases of the digestion and measured as described by Nissen et al. [ 37 ]. DH data were corrected with the data obtained in the control digestion. Determination of ACE-I activity ACE-I activity was determined by the method of Cushman and Cheung [ 38 ]. Accordingly, 20 µL LP of in vitro digested cheese was added to 100 µL 5 mmol L − 1 hippuryl-histidy-leucine (HHL, Sigma) substrate solution (dissolved in 0.1 mol L − 1 sodium borate buffer solution containing 0.3 M NaCl, pH 8.3). After adding 40 µL of 0.1 U/mL ACE (from rabbit lung, Sigma) reagent to this suspension, the reaction mixture was further incubated at 37 ºC for 30 min and finished with 150 µL of 1 mol L − 1 HCl. Then 800 µL of the organic phase was transferred to a glass tube and then dried out by heating at 95°C for 20 min to evaporate ethyl acetate. The residue hippuric acid was dissolved in 1 mL H 2 O and absorbance was measured at a wavelength of 228 nm (Shimadzu, UV-1800 UV–vis, Japan). The result was expressed as ACE-I activity %, calculated using the equation: (A − B)/(A − C) × 100, where A is the absorbance without sample, B is the absorbance without ACE, and C is absorbance in the presence of both ACE and sample. Determination of antioxidant activity The antioxidant capacity of the LP extracted from in vitro digested cheese was determined according to ABTS radical scavenging and CUPRAC methods. Trolox equivalent antioxidant capacity (TEAC) of the cheeses according to the ABTS decolorization assay was performed by mixing 10, 20, and 30 µL of samples and 1000 µL of ABTS solution in quartz micro cuvette, respectively. After 6 min incubation period, the absorbance of the solution was measured at 734 nm using a spectrophotometer (Shimadzu, UV-1800 UV–vis, Japan). TEAC values were reported as mM of Trolox equivalents per 100 mg cheese [ 39 ]. For CUPRAC method; 70 µL copper (II) solution (10 mM), 70 µL neocuproin solution (7.5 mM) and 70 µL ammonium acetate buffer (1 M) were added into the microplate wells, respectively, and 7 µL sample was added followed by 70 µL distilled water. The microplate was shaken well for 10 s, sealed and kept at room conditions for 30 min. At the end of 30 min, absorbance was measured at a wavelength of 450 nm (NanoQuant Infinite M200, Tecan, Switzerland) against the blank. Antioxidant activities were reported as mM Trolox g − 1 cheese [ 40 ]. Statistical analysis Cheeses were compared using general linear model ANOVA at each ripening day to determine effects of cheesemaking process (heating process, starter culture addition and coagulant origin), and one-way ANOVA to evaluate effect of ripening time for each cheese. Comparison of means was carried out by using Tukey’s or Welch (for the non-parametric data) test. Data were obtained from repeated analysis and evaluated by Minitab 18.1 (Minitab 2017) software. Results were expressed as mean ± standard error to indicate the precision of the sample mean and account for variability in the data. Results and discussion Chemical composition of cheeses Total solids, fat/TS, protein/TS and salt/M of the cheeses are presented in Table 2 . TS of cheeses were ranged between 38.56–51.69%. The interaction of heat treatment and starter culture affected the TS of the cheeses (P ≤ 0.01); however, use of plant or animal origin coagulating enzyme did not significantly affect (P > 0.01). TS ​​of the cheeses increased significantly during ripening (P ≤ 0.01), except HS cheeses, and the values changed approximately 7% in raw milk cheeses and 12% in HF cheeses. Total solid of cheese is directly affected by the composition of milk, cheese production technique and ripening condition. The salt moves towards the center of the cheese mass and disperses in the structure of the cheese during salting process. The dispersion of salt continues throughout ripening until the salt is completely homogeneously distributed in the cheese mass. In a study reporting similar results to this study; Uzkuç et al. [ 41 ] reported that there was no difference between the TS ​​of goat cheeses produced from milk coagulated with C. cardunculus protease or animal rennet, and TS ​​increased during ripening. The changes in TS ​​during 90 days ripening were attributed to salt migration, which led to whey or water release from the cheese[ 41 ]. The fact that RF, RS and HF cheeses ripened for 90 days have higher TS than fresh cheeses can be attributed to similar reason. Similar increase in TS result also observed in Serbian traditional goat cheese during ripening [ 42 ]. The fact that the TS ​​of HS1 and HS2 cheeses did not change during the ripening period can be explained by referring to the pH, titratable acidity results presented on the previous publication of the cheeses in this study. Accordingly, this may be due to the fact that HS cheeses reached their final pH (4.52) and titratable acidity (1.96) values ​​on the 1st day of ripening and the rest of the ripening period did not show the ripening process characteristics observed in the other cheese groups [ 10 ]. The role of pH in cheese texture is particularly important because changes in pH are related directly to chemical changes in the protein network of the cheese [ 10 , 43 ]. Barac et al. [ 31 ] found significantly increased TS during 50 days of ripening in traditional Serbian goat cheeses produced by heat treatment at 90° C for 10 min, ranging from 44.88–49.45%, which are comparable to the cheeses produced from heat-treated milk in the present study. However, Kocak et al. [ 44 ] reported significant increase in moisture (decrease in TS) content of goat cheese ripened in brine during storage, attributing the change to the movement of water from the brine to the cheese curd as a consequence of the low salt concentration in the brine (< 20%). Unlike the results, in this study, no decrease in dry matter was observed because the cheeses were not stored in brine during the ripening period. The TS results determined in this research during ripening were also comparable to the TS reported for goat milk cheeses in other studies [ 45 , 46 ]. The fat/TS ratios of the cheeses were ranged from 47.70 to 56.89% (Table 2 ). Heat treatment, starter culture addition, plant coagulant or their interaction did not affect significantly the fat/TS of the cheeses (P > 0.01). Fat/TS of HF cheeses increased at the end of the ripening (P ≤ 0.01), this change may be attributed to a high rate of TS change in ripened cheeses compared to the fresh cheeses. In fresh soft goat cheeses produced from pasteurized milk using thermophilic and mesophilic starter cultures, fat/TS ratios were found between 49.8 and 54.2% during 60 days ripening [ 47 ], similar to the fat/TS finding in this study. The fat/TS [ 48 , 49 ] and protein/TS contents of the different goat cheeses are generally comparable with the result in the present study, although there are minor differences due to the composition of raw milk and cheese-making procedures. The effect of interaction between heat treatment and starter culture addition on the protein/TS results of the cheeses was significant only 60th and 90th days of ripening (P ≤ 0.01). The protein/TS ratios of cheeses ranged between 45.14 and 49.33% on the 1st day of ripening, while the values were determined between 38.20 and 42.42% on the 90th day of ripening. Popovic-Vranjes et al.[ 50 ] reported fat/TS ratios between 38.42 and 44.78% in goat cheeses, which were similar with the present study. The protein/TS ratios of cheeses decreased during ripening in all cheeses (P ≤ 0.01) (Table 2 ). Protein/TS findings in the present study were similar to the findings presented by Miloradovic et al.[ 51 ] for high heat-treated fresh goat (Saanen) cheeses (46.87–48.85%). However, lower protein/TS ratios than the present study were reported by Barac et al.[ 52 ] for white brined goat cheese and by Álvarez and Frenso[ 53 ] for all age Palmero cheese (26.98–31.37%). The higher protein/TS results than different goat cheeses [ 52 , 53 ] may be attributed to the milk source (Palmero Goat) and cheesemaking techniques (different salting and ripening methods). The salt/M ratio of the cheeses were ranged between 4.76–6.54% (Table 2 ). The interaction effect of heat treatment and starter culture addition on the salt/M of the cheeses for day 1 was significant (P ≤ 0.01). HF cheeses (5.77–6.54%) differed from the HS cheeses (5.13–5.40) in terms of salt/M on the 1st day of ripening (P ≤ 0.01). The salt/M of the cheeses did not change significantly during ripening (P > 0.01) (Table 2 ). A study on goat milk cheeses, which produced combination of dry (3%) and brine (8%) salting indicated salt/M contents from 1.76 to 5.67% during 40 days of ripening [ 51 ]. Salt/M findings in the present study is similar for 30–40 days ripened cheeses, but different for fresh cheeses found by Miloradovic et al. [ 51 ], which can be attributed to the difference in salting technique and salting time. In addition, Kocak et al. [ 44 ] also found similar salt/M ratios with this study for brined goat cheeses on ripening days but reported significantly increasing salt/M results during ripening. They expounded that the increase in salt/M content was expected during ripening due to salt diffusion from brine to cheese increases [ 44 ]. Therefore, statistically insignificant salt/M changes during ripening in this study may be attributed to the fact that cheeses were not ripened in brine. Hydrolysis degree (DH) Proteolysis is the breakdown of large and complex proteins into smaller and simpler peptides. In cheese, the presence of bioactive peptides is the result of a sensitive equilibrium between their release and their degradation by the activity of lactic acid bacteria proteinases and peptidases during cheese ripening [ 54 ]. Proteolysis results in the cheeses during cheese production and ripening processes were presented in our previous study [ 10 ]. Accordingly, it was reported in detail that the water-soluble nitrogen in total nitrogen (WSN/TN) and total free amino acids (TFAA) results, which are some indicator analyses for proteolysis, were significantly affected by the interaction of heat treatment and starter culture addition. Moreover, WSN/TN and TFAA underwent noticeable increases during the ripening process of all cheeses. It was reported that HF ​​cheeses had limited proteolysis according to WSN/TN and TFAA, while HS group cheeses reaching values 30.26 (% in TN) and 12.72 (mg leu 100 g − 1 ) in the end of the ripening. In addition, RP-HPLC peptide profiles were observed in the water-soluble fractions of the cheeses and Urea-PAGE analyses were observed in the water-insoluble nitrogen fractions, and the results were in accordance with the WSN/TN and TFAA results [ 10 ]. Enzymatic hydrolysis improves the functional properties of dietary protein by converting it into bioactive peptides while preserving its nutritional value. The digestion process has the noteworthy potential that proteolytic digestive enzymes can release bioactive peptides from goat milk proteins [ 55 ]. The hydrolysis of goat cheese protein and peptides with the in vitro digestion was evaluated consulting the OPA assay based on the measurement in free NH 2 groups. Figure 1 shows the resulting DH, total of salivary, peptic and pancreatic digestion, relative to the control condition. Heat treatment, starter culture addition, plant coagulant or their interaction did not change significantly the DH of the cheeses (P > 0.01). After 2 min salivary, 2 h peptic and 2 h pancreatic of digestion, the average DH of cheeses was 86,43%. HF2 cheese had the lowest DH (81.38%) and HS2 cheese had the highest DH value (91.09%). This result can be attributed to the relatively low moisture content and/or higher salt concentration of HF2 compared to HS2. Higher moisture content can lead to greater salt solubility, and the salt concentration in cheese affects protein digestibility. Increased salt levels may decrease protein solubility [ 56 ], which can make protein digestion more difficult. The degree of hydrolysis fluctuated during ripening for in cheeses and no general trend was found for the release of free NH 2 groups by in vitro digestion in cheeses on different ripening days. However, DH did not change significantly during ripening (P > 0.01). In a study examining the amount of peptide released from Ras cheese through in vitro digestion, it was reported that, similar to our study, there was no difference between cheeses ripened for different periods [ 57 ]. ACE-I activity ACE-I activity of LPs extracted from in vitro digested cheeses that contains the potential bioactive peptides consisting of 2–20 amino acids in cheese were presented in Table 3 . The combination of heat treatment and starter culture addition significantly affected the ACE-I activity of the cheeses on all ripening days (P ≤ 0.01). Cheeses were divided into four groups according to their ACE-I activities on the 1st day of ripening. Initially, HF cheeses had the lowest (13.15–15.52%) and HS cheeses had the highest (76.65–77.94%) ACE-I activity ​​(Table 3 ). HF cheeses also had lower ACE-I activities ​​than other cheeses on 1st, 30th and 60th days of ripening while reaching similar ACE-I activity ​​as RS and HS cheeses on 90th day. 30, 60 and 90 days ripened RF cheeses had higher ACE-I activity ​​than the cheeses produced from heat-treated milk. However, it was found that cheeses with starter culture added had similar ACE-I activity ​​on the 30th, 60th and 90th days of ripening, regardless of the heat treatment and coagulant origin. The microflora of cheeses can affect the formation of bioactive peptides [ 58 ]. It has been reported that raw milk cheeses show higher ACE inhibitor activity than cheeses produced from pasteurized milk, depending on the proteolytic enzymes of microorganisms in the natural microflora of milk [ 59 ]. In addition, starter cultures break down the ACE-I peptides in cheeses into peptides and amino acids with lower or higher activity [ 60 ], and Lactobacillus helveticus is generally more effective than Lactococcus lactis in the release of ACE-I peptides in cheese [ 61 ]. Saremnezhad et al. [ 62 ] reported the high ACE-I effect of the Lighvan cheese made from 100% raw goat's milk compared the cow milk cheeses, and they attributed to the amino acid sequence of the peptide fragments from goat's milk and the proteolytic power of the native microbiota of this type of milk. It has been reported that in vitro digestion reduces the ACE-I activity in the water-soluble fractions of the cheeses, and this may be attributed to the loss of activity of ACE-I peptides as a result of hydrolysis by digestive enzymes [ 27 , 31 , 63 ]. Ripening time had significantly affected ACE-I activity of cheeses (P ≤ 0.01). Increases and decreases were observed in the ACE-I activity of raw milk cheeses during ripening, and the highest ACE-I activity in cheeses were determined on the 30th and 60th days of ripening in RF cheeses (average values were 76.17 and 68.52%, respectively) and on the 30th days of ripening in RS cheeses (average value was 68.72%). In a study, the highest ACE-I activities ​​were determined in 60 days-ripened brined goat cheese produced using starter culture as 76.15% and 51.95%, respectively [ 44 ]. Researchers reported increases and decreases in the ACE-I activity of cheeses during ripening [ 64 – 66 ]. Kocak et al. [ 44 ] determined a decrease in the ACE-I activity of goat cheeses after the 60th day of ripening and attributed the decrease to the breakdown of active peptides into inactive form due to proteolysis. The average ACE-I activity of HF cheeses increased from 14.34–47.59% during ripening, however, average ACE-I activity of HS cheeses decreased from 77.30–44.56% (Table 3 ). Özcan Yardım and Durak [ 29 ] reported higher in vitro ACE-I activity in goat cheese on 90th ripening day compared to sheep, cow and Van herby cheese. The lower ACE-I activity found in their goat cheese (10.44%) compared to all findings in our study may be due to differences in their digestion procedure for the water-soluble extract of cheeses. Table 3 ACE-I activities of goat cheeses (%) Day Cheese RF1 RF2 RS1 RS2 HF1 HF2 HS1 HS2 1 55.40 ± 3.20 Bb 52.65 ± 1.86 Bb 40.14 ± 1.82 Bc 38.95 ± 3.66 Bc 15.52 ± 3.37 Bd 13.15 ± 2.92 Bd 76.65 ± 4.39 Aa 77.94 ± 2.34 Aa 30 76.91 ± 1.76 Aa 75.43 ± 2.48 Aa 70.23 ± 5.48 Aab 67.21 ± 5.20 Aab 18.10 ± 0.81 Bc 20.24 ± 2.16 Bc 56.82 ± 4.38 ABb 60.66 ± 5.56 ABb 60 68.45 ± 3.31 ABa 68.60 ± 1.44 ABa 57.44 ± 2.20 ABb 53.81 ± 4.28 ABb 24.99 ± 4.30 Bc 23.63 ± 2.93 Bc 47.32 ± 4.92 Bb 53.25 ± 2.45 Bb 90 59.05 ± 5.30 ABa 61.57 ± 5.66 ABa 52.37 ± 2.28 ABab 50.55 ± 2.41 ABab 49.57 ± 3.35 Ab 45.61 ± 4.15 Ab 45.72 ± 1.89 Bb 43.40 ± 2.35 Bb Values are expressed as mean ± standard error.Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P ≤ 0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter. Researchers reported that the ACE-I activity of cheeses affected by milk pretreatment, starter cultures, scalding conditions, and ripening [ 63 , 67 ]. Torres-Llanez et al. [ 68 ] reported that ACE inhibitor peptides in unripened cheeses were formed by the proteolytic effect of starter cultures. Thus, the starter culture used in the production of HS cheeses provided the formation of peptides with high ACE-I activity in the cheese on the 1st day of ripening. The relatively low ACE-I activity determined in the first 60 days of ripening in the HF cheeses may be attributed to the lower proteolysis level in the absence of starter culture, thus lower bioactive peptide formation, which may be associated with the limited proteolytic activity of the residual milk-clotting enzyme. In addition, increase in ACE-I activity of HF cheeses on the 90th day of ripening might be attributed to the presence of lactococci and lactobacilli in those cheeses as a result of contamination starter or heat tolerant raw milk microflora explained in the previous study [ 10 ]. Antioxidant activity The antioxidant activity results of the LPs determined by the ABTS radical scavenging method are presented in Table 4 . The effects of heat treatment and starter culture addition on the antioxidant activities of cheeses on the 1st and 30th days of ripening was found to be significant (P ≤ 0.01). On the 1st day of ripening, RF1 and RF2 cheeses had the highest antioxidant activity (69.02–65.46 mM Trolox 100 mg − 1 cheese), while HF1 and HF2 cheeses had the lowest antioxidant activity (45.95–46.75 mM Trolox 100 mg − 1 cheese). In the cheeses ripened for 30 days, raw milk cheeses (RF and RS) had higher antioxidant activity than cheeses produced from heat-treated milk. The antioxidant activities of starter free cheeses (RF1-RF2 and HF1-HF2) changed significantly during ripening (P ≤ 0.01). Saremnezhad et al. [ 62 ] reported the highest ABTS inhibition activity (61.26%) in the 90-day ripened Lighvan cheese made from 100% raw goat's milk, and they attributed the activity to structure of goat milk protein-forming amino acids and the proteolytic power of the enzymes present in the natural microflora of the milk. Antioxidant peptides are mostly released from caseins under the influence of starter and non-starter lactic acid bacteria [ 52 ]. Related to this statement, it was determined that natural microflora contributed to antioxidant activities in cheeses produced from raw milk among the cheeses stored for 30 days, and in addition, it was found that antioxidant capacities were higher among cheeses produced from heat-treated milk on the 1st and 30th days of ripening, depending on the starter culture activity added into HS1 and HS2 cheese milk. Table 4 ABTS antioxidant activities of goat cheeses (mM Trolox 100 mg − 1 cheese). Day Cheese RF1 RF2 RS1 RS2 HF1 HF2 HS1 HS2 1 69.02 ± 0.47 Aa 65.46 ± 2.11 Aa 58.98 ± 1.45 b 57.74 ± 0.41 b 45.95 ± 0.44 Cc 46.75 ± 1.21 Bc 59.18 ± 3.46 b 61.76 ± 1.09 b 30 66.58 ± 1.07 Aa 57.73 ± 0.96 Ba 63.89 ± 0.82 c 59.51 ± 0.48 c 47.82 ± 1.01 BCc 49.13 ± 1.32 Bc 57.06 ± 1.97 b 59.36 ± 1.43 b 60 56.42 ± 2.83 B 55.89 ± 0.80 B 61.05 ± 3.64 61.21 ± 2.60 55.98 ± 3.48 AB 54.13 ± 2.29 AB 59.08 ± 0.72 58.03 ± 1.23 90 63.00 ± 2.03 AB 59.81 ± 1.23 AB 62.90 ± 1.18 60.48 ± 2.41 58.90 ± 2.15 A 59.70 ± 2.79 A 59.64 ± 4.07 57.56 ± 2.56 Values are expressed as mean ± standard error. Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P ≤ 0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter. CUPRAC results of LPs during ripening were presented in Table 5 . The interaction of heat treatment and starter culture addition on the antioxidant activities of the cheeses in terms of CUPRAC was found to be significant (P ≤ 0.01). The lowest antioxidant activities were determined as 9.01–9.65 on the 1st day of ripening and 9.15–9.41 mM trolox g − 1 cheese on the 30th day of ripening in HF cheeses, while the highest antioxidant activities ​​were determined as 16.88–17.52 on the 1st day of ripening and 16.34–15.88 mM trolox g − 1 cheese on the 30th day of ripening in RF cheeses, respectively. The antioxidant activities of raw milk cheeses decreased significantly during ripening (P ≤ 0.01) and the approximate average decrease rates were 21% in RF cheeses and 14% in RS cheeses. It can be interpreted that the enzymes originated from raw milk microflora released more low molecular weight peptides with antioxidant activity during the production of raw milk goat cheese. Accordingly, it was found that HF cheeses that were heat-treated but did not contain starter culture had lower antioxidant activity ​​than the other cheeses on the 1st, 30th and 60th days of ripening, and had similar results ​​with HS cheeses at the end of 90th day of ripening (Table 5 ). Table 5 CUPRAC antioxidant activities of goat cheeses (mM Trolox g − 1 cheese). Day Cheese RF1 RF2 RS1 RS2 HF1 HF2 HS1 HS2 1 16.88 ± 0.18 Aa 17.52 ± 0.34 Aa 13.94 ± 0.36 Ab 14.00 ± 0.02 Ab 9.01 ± 0.34 d 9.65 ± 0.30 d 11.15 ± 0.28 c 10.71 ± 0.32 c 30 16.34 ± 0.36 ABa 15.88 ± 0.22 ABa 13.70 ± 0.12 Ab 13.96 ± 0.14 Ab 9.15 ± 0.28 d 9.41 ± 0.14 d 10.85 ± 0.02 c 10.63 ± 0.16 c 60 14.74 ± 0.36 BCa 14.40 ± 0.38 BCa 12.63 ± 0.20 ABb 11.73 ± 0.26 Bb 8.85 ± 0.34 c 10.17 ± 0.22 c 10.81 ± 0.02 b 11.53 ± 0.06 b 90 13.06 ± 0.24 Ca 13.98 ± 0.20 Ca 11.81 ± 0.18 Bb 12.13 ± 0.34 Bb 8.99 ± 0.28 c 10.07 ± 0.08 c 10.85 ± 0.02 bc 10.09 ± 0.30 bc Values are expressed as mean ± standard error. Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P ≤ 0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter. A few research showed that peptide formation in cheese production increases the antioxidant capacity of fresh cheeses, and the same quality increases during the ripening process [ 32 , 69 ]. Revilla et al. [ 70 ] found that the highest antioxidant activity (ABTS) in goat cheeses was determined on the 90th day of ripening, that producing cheese from milk obtained in different seasons differentiates the antioxidant activity, especially in fresh cheeses, and that summer cheeses have lower hydrophobic:hydrophilic ratios than winter cheeses, which results in strong proteolysis. Accordingly, they reported that fresh cheeses produced in summer months had higher antioxidant capacity. In a study investigating the antioxidant activities of white cheeses produced from cow and goat milk without adding starter culture, the antioxidant capacities of the water-soluble fractions of the goat cheeses, before and after in vitro digestion, were 159.97 and 152.41 mM Trolox kg − 1 , respectively, and it was found to be 25.33 and 81.43 mM Trolox kg − 1 in the insoluble fractions, respectively [ 31 ]. In that study, it was stated that the antioxidant activities of goat cheese protein fractions increased significantly with in vitro digestion, and the TEAC ​​determined on the 50th day of ripening were higher than the results ​​of less stored cheeses. It was reported that in vitro digestion enhanced the antioxidant activity of 2-month-old fresh Kradi cheeses [ 71 ]. In a study investigating the in vitro antioxidant activities (ABTS and FRAP) of water-soluble fractions (< 3 kDa) of ripened cow, goat and Ezine cheeses produced by the same method, Turan and Durak [ 27 ] reported that goat cheese had a higher antioxidant capacity than other cheeses. In addition, the antioxidant activity differences between cheeses are related to the types of milk used in the production of cheese types as well as the number of peptides formed by the hydrolysis of the proteins of these milks to form different peptides during the proteolysis that occurs during the ripening of the cheeses. Öztürk and Akın [ 72 ] found that Tulum Goat cheese, produced by fermenting goat milk with its natural microflora and without applying heat treatment to the milk, had a higher antioxidant activity than cow Tulum cheese produced under the same conditions, according to the DPPH method, and this was due to the higher hydrophilic peptide content in goat milk Tulum cheese. Conclusion In this study, the bioaccessibility was assessed in the low molecular weight peptides of in vitro digested the goat cheeses. Cheesemaking factors investigated in the study, heat treatment, starter culture addition and ripening, affected the bioaccessibility of goat cheese regardless of the source of milk coagulation enzyme used in production. Therefore, the goat cheeses showed ACE-I and antioxidant activities. It has been determined that in cheeses produced from heat-treated milk with the presence of starter culture, ACE-I activity decrease during ripening due to proteolytic activity of starter culture, but on the contrary, results increased in the absence of starter culture. The antioxidant activity was tested with two different assays ABTS and CUPRAC. For both tests, the free radical scavenging activity changed positively or remained unchanged following in vitro digestion. The interaction of heat treatment and starter culture addition was affected the ABTS antioxidant activities of LP after in vitro digestion of cheeses on the 1st and 30th days of ripening. ABTS values of RF group decreased, but the antioxidant properties of HF group increased during ripening while the antioxidant capacities of cheeses produced from starter culture added milk did not change. It was found that the CUPRAC values ​​of raw milk cheeses decreased with ripening, but the values ​​of heat-treated milk cheeses did not change. It was concluded that cheeses produced using plant-derived and animal coagulants were generally similar in terms of bioaccessibility. Accordingly, it has been shown that wild artichoke ( C. cardunculus ) protease can be used as an alternative to produce goat cheese to obtain similar ACE-I and antioxidant activities. The findings of this work confirm that all age cheeses produced from raw milk without starter addition and fresh cheeses produced from heat-treated starter added milk, present over 50% ACE-I activity and can be a source of peptides with antihypertensive health-promoting properties. According to the results obtained in this study, raw goat milk cheeses have the potential to reduce oxidative damage by presenting bioactive peptides which have the antioxidant properties. To fully understand the health effects, it is necessary to conduct in vivo studies. Further studies are required to put forward the new functional properties of goat cheeses and proteomic research to sequence peptides correlated to bioaccessibility properties and characterization bioactive peptides. Declarations Acknowledgments This work is the part of PhD study of Hasan UZKUÇ under the supervision of Prof. Dr. Yonca KARAGÜL YÜCEER at the Department of Food Engineering, Faculty of Engineering, Çanakkale Onsekiz Mart University, Türkiye. Funding This work is supported by the Scientific Research Coordination Unit of Çanakkale Onsekiz Mart University with grant number: FBA-2021-3585. Author contributions Hasan UZKUÇ : Conceptualization, writing – original draft, software, formal analysis, data curation, visualization. Yonca KARAGÜL YÜCEER : Conceptualization, resources, writing – review and editing, supervision. Conflict of Interest The authors declare that they have no conflict of interest Compliance with ethics requirements This article does not contain any studies with human or animal subjects. References TUIK (2024) Turkish Statistical Institute- Livestock Statistics. In: https://www.tuik.gov.tr FAO (2024) Food and Agriculture Organization of the United Nations. 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Food Sci Nutr. https://doi.org/10.1002/fsn3.4543 Lignitto L, Cavatorta V, Balzan S et al (2010) Angiotensin-converting enzyme inhibitory activity of water-soluble extracts of Asiago d’allevo cheese. Int Dairy J 20:11–17. https://doi.org/10.1016/j.idairyj.2009.07.001 Gómez-Ruiz JÁ, Ramos M, Recio I (2002) Angiotensin-converting enzyme-inhibitory peptides in Manchego cheeses manufactured with different starter cultures. Int Dairy J 12:697–706. https://doi.org/10.1016/S0958-6946(02)00059-6 Meira SMM, Daroit DJ, Helfer VE et al (2012) Bioactive peptides in water-soluble extracts of ovine cheeses from Southern Brazil and Uruguay. Food Res Int 48:322–329. https://doi.org/10.1016/j.foodres.2012.05.009 Ryhänen EL, Pihlanto-Leppälä A, Pahkala E (2001) A new type of ripened, low-fat cheese with bioactive properties. Int Dairy J 11:441–447. https://doi.org/10.1016/S0958-6946(01)00079-6 Pisanu S, Pagnozzi D, Pes M et al (2015) Differences in the peptide profile of raw and pasteurised ovine milk cheese and implications for its bioactive potential. Int Dairy J 42:26–33. https://doi.org/10.1016/j.idairyj.2014.10.007 Torres-Llanez MJ, González-Córdova AF, Hernandez-Mendoza A et al (2011) Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese. J Dairy Sci 94:3794–3800. https://doi.org/10.3168/jds.2011-4237 Moreno-Fernandez J, Alférez MJM, López-Aliaga I, Diaz-Castro J (2019) Protective effects of fermented goat milk on genomic stability, oxidative stress and inflammatory signalling in testis during anaemia recovery. Sci Rep 9(1):6881. https://doi.org/10.1038/s41598-018-37649-6 Revilla I, González-Martín MII, Vivar-Quintana AMM et al (2016) Antioxidant capacity of different cheeses: Affecting factors and prediction by near infrared spectroscopy. J Dairy Sci 99:5074–5082. https://doi.org/10.3168/jds.2015-10564 Mushtaq M, Gani A, Shetty PH et al (2015) Himalayan cheese (Kalari/kradi): Effect of different storage temperatures on its physicochemical, microbiological and antioxidant properties. LWT 63:837–845. https://doi.org/10.1016/j.lwt.2015.04.054 Öztürk Hİ, Akin N (2018) Comparison of some functionalities of water soluble peptides derived from Turkish cow and goat milk Tulum cheeses during ripening. Food Sci Technol (Brazil) 38:674–682. https://doi.org/10.1590/1678-457x.11917 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6542067","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453448760,"identity":"d6fd1d1d-a5f9-4757-bcbc-6843868c4907","order_by":0,"name":"HASAN UZKUÇ","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":false,"prefix":"","firstName":"HASAN","middleName":"","lastName":"UZKUÇ","suffix":""},{"id":453448761,"identity":"d16dd251-aba0-48b7-8ceb-f3ccbeb4f0e3","order_by":1,"name":"YONCA KARAGÜL YÜCEER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYJADxgdQhgHRWphhSoF0AnFa2CSI0mJwvPfgh59th+X52XufVfyo2JbYwN68TYLxxz3cWs6cS5bsbTtsOLPnuNnNnjO3Ext4jpVJMCQU49ZyI8eMgbftcILBjTS224xtQC0SOWZALbhdBtLC+Beoxf7+M7ZisBb5N4S1MINtkWBjY4bYwoNfi+SZM8bSMufSDWecSWOWBPrFuI0nrdgiIQ23Fr7jPYYf35RZy/O3H2P88KPitmw/++GNNz7Y4NaicABIMLIhiYDZuDUwMMg3gMg/eFSMglEwCkbBKAAAXUhTnM6AF+4AAAAASUVORK5CYII=","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":true,"prefix":"","firstName":"YONCA","middleName":"KARAGÜL","lastName":"YÜCEER","suffix":""}],"badges":[],"createdAt":"2025-04-27 18:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6542067/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6542067/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82296331,"identity":"c2299e53-c584-44e6-a065-27e66c020378","added_by":"auto","created_at":"2025-05-08 19:42:10","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136795,"visible":true,"origin":"","legend":"\u003cp\u003eHydrolysis degree (DH) of cheeses.\u003c/p\u003e\n\u003cp\u003eValues are expressed as mean ± standard error. Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6542067/v1/c777b89f4fcfecc59f515760.jpeg"},{"id":82296695,"identity":"86fe0c61-05e6-4aa9-a3cb-47663ff85cb9","added_by":"auto","created_at":"2025-05-08 19:50:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1078629,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6542067/v1/202199f5-fbbc-43aa-b116-1194162093fe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of heat treatment, starter culture, plant coagulant and ripening on in vitro ACE inhibitory and antioxidant activity of goat cheese","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThere are 10.3\u0026nbsp;million goats in Turkey, which has 1% of the world's goat population, and 2.5% of the total milk production (21.5\u0026nbsp;million tons) is goat milk [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. World goat milk production, which was 10\u0026nbsp;million tons in 2010, doubled in 2020 and reached around 20.6\u0026nbsp;million tons [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This increase in the amount of goat milk production can be attributed to the increasing world population, the increasing awareness of consumers and interest in functional foods, and the rapid increase in the demand for goat milk. In recent years, researchers have reported that consumers have shown interest in goat milk and its products due to their high nutritional values ​​and the presence of bioactive components important for a healthy life [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Goat milk stands out with its casein micelles have smaller size, higher solubility, lower sedimentation rate, lower heat stability, and higher calcium and phosphorus content than cow milk [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and also the fact that goat milk contains lower concentrations of α\u003csub\u003es1\u003c/sub\u003e-casein and higher concentrations of K-casein and β-casein contributes to its easy digestibility and lower allergenicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Goat milk proteins, due to the extensive genetic polymorphism of goats, might have different amino acid composition, which can lead to significant differences in the sensory, digestibility and bioactive properties of goat milk products [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGoat milk is used in various products such as fermented food, functional foods, and cosmetics. Certainly, one of the most effective utilization and ripening methods of goat milk is cheese making [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cheeses are not only known for their rich content of high- quality proteins, lipids, vitamins, and essential minerals, but they also harbor bioactive components, such as bioactive peptides [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These peptides support various body functions, but they are initially inactive within the protein structure and are released or activated through proteolysis processes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Hydrolysis processes of the goat milk proteins have been reported to produce peptides with antihypertensive, antioxidant, hypoglycemic, anti-inflammatory, antimicrobial, opioid or immunomodulatory activities [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn cheeses, bioactive peptides are released by the action of the milk coagulation enzyme, as well as by the activity of starter and non-starter lactic acid bacteria and act as potential antioxidants and ACE inhibitors[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Bioactive peptides which have the antioxidant properties reduce oxidative damage by reacting with free radicals or inhibiting oxidative reactions. Free radicals are highly unstable molecules that can easily interact with other compounds and cause oxidative damage in the body, which has been linked to various diseases such as arthritis, diabetes, and cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Otherwise, antihypertensive activity of peptides is given through the inhibition of ACE, preventing the conversion of angiotensin I to angiotensin II, which induces the release of aldosterone, increasing sodium concentration and blood pressure; and inactivating the vasodilator bradykinin, protecting the body against cardiovascular disease [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eACE-I and antioxidant activity have been reported to be strongly associated with ongoing proteolysis, bioactive and non-bioactive peptides during cheese production and ripening [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been stated that the milk source used in production, the technological processes applied in production, and the strain selection of bacteria involved in proteolysis were effective on the enzyme activation of bioactive peptides in cheese [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Considering the diversity in production parameters, many bioactive peptides originating from milk proteins can be obtained through cheese production, and by revealing the effects of cheese production parameters, the production of bioavailable cheeses can be optimized without using additional additives. On the other hand, the functional properties of peptides released during the cheese production process depend on the changes the peptides undergo during gastrointestinal digestion and subsequent absorption [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since the presence of bioactive peptides in foods does not guarantee the physiological effect of these peptides after digestion, \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies are required to prove the potential health benefits resulting from cheese consumption.\u003c/p\u003e \u003cp\u003eSince the last 20 years, very few studies have focused on the investigation of goat milk proteins and their fractions, especially for the presence of bioactive peptides. For this reason, it is thought that there is still undiscovered information in goat milk and its products, mainly in the field of bioactive peptides [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although a large number of peptides of milk protein origin have been identified, it has been reported that very few of these peptides show proven antihypertensive properties under \u003cem\u003ein vitro\u003c/em\u003e conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Goat milk and kefir displayed about 7-fold antioxidant activity after digestion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, it is important to reveal the bioaccessibility of peptides by conducting research on the antioxidant and ACE-I activities of goat cheeses under \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal digestion conditions.\u003c/p\u003e \u003cp\u003eIn recent decade a few studies undertaken \u003cem\u003ein vitro\u003c/em\u003e ACE-I and antioxidant activity of goat cheeses [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, some studies investigated ACE-I and antioxidant potential of goat cheese in low (\u0026lt;\u0026thinsp;3 kDa) molecule weight fractions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This study aimed to contribute to the literature by introducing the combined effects of heat treatment, starter culture addition, plant coagulant and ripening processes on the \u003cem\u003ein vitro\u003c/em\u003e ACE-I and antioxidant activities of semi-hard goat cheese\u0026thinsp;\u0026lt;\u0026thinsp;3 kDa peptide fractions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCheesemaking\u003c/h2\u003e \u003cp\u003eRaw and 90 \u0026ordm;C 10 min heat-treated Turkish Saanen goat\u0026rsquo;s milk (İmbroz Mutlu Ke\u0026ccedil;iler \u0026Ccedil;iftliği, G\u0026ouml;k\u0026ccedil;eada, T\u0026uuml;rkiye) was used to produce cheeses without adding starter culture and with adding a mesophilic-thermophilic starter culture (CHOOZIT MA 4002, Danisco, Denmark). Goat milk was coagulated with a plant sourced rennet originated from \u003cem\u003eCynara cardunculus L.\u003c/em\u003e (Coagulante Vegetal, Pontevedra, Spain) or a calf rennet (Animal Rennet, Rumeli Maya, İstanbul T\u0026uuml;rkiye). Cheese ripening process caried out at 6\u0026ndash;8 \u0026ordm;C for 90 days. Cheeses were produced from a single batch of goat milk in duplicate trials, as detailed in our previous study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and the cheesemaking parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The cheeses were analyzed for bioaccessibility properties at 1st, 30th, 60th and 90th days of ripening. Cheeses were coded by using abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTechnological parameters of goat cheeses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterial \u0026amp; Process\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCheese Milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTurkish Saanen goat milk (120 L), 6.49 pH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.1% total solids, 3.5% protein, 3.5% fat\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeat treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo heat (R) or 90\u0026deg;C 10 min (H)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCooling temprature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarter culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eWithout starter\u003c/em\u003e (RF, HF)*\u003c/p\u003e \u003cp\u003e\u003cem\u003eor\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLactococcus lactis\u003c/em\u003e subsp. \u003cem\u003elactis\u0026thinsp;+\u0026thinsp;Lactococcus lactis\u003c/em\u003e subsp. \u003cem\u003ecremoris\u003c/em\u003e +\u003c/p\u003e \u003cp\u003e\u003cem\u003eLactococcus lactis\u003c/em\u003e subsp. \u003cem\u003elactis biovar. diacetylactis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eStreptococcus thermophilus\u003c/em\u003e (RS, HS)*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.04%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaturation of milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u0026ndash;45 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoagulant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCynara cardunculus L\u003c/em\u003e. protease (100 IMCU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (RF1, RS1, HF1, HS1)*\u003c/p\u003e \u003cp\u003eor\u003c/p\u003e \u003cp\u003eCalf rennet (180 IMCU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (RF2, RS2, HF2, HS2)*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClotting time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCutting of the curd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 cm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheying-off\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePressing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3100 Pa for 30 min\u0026thinsp;+\u0026thinsp;6200 Pa for 60 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaalting into brine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12% NaCl-12-14 h at 27.5\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCutting of the cheese\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 equal parts\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePacking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlastic vaccum packing material\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRipening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u0026ndash;8\u0026deg;C \u0026ndash; 90 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cem\u003e*R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet\u003c/em\u003e, codes indicate process factor in cheesemaking.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCheese analyses\u003c/h3\u003e\n\u003cp\u003eCheese samples were analyzed for total solids (TS), protein (expressed as protein in TS (protein/TS)) and salt (expressed as salt in moisture (Salt/M)) according to [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and fat (expressed as fat in TS (fat/TS)) according to [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] at 1st, 30th, 60th and 90th days of ripening. Chemical composition of fresh cheeses (day 1) was adapted from Uzku\u0026ccedil; and Karag\u0026uuml;l Y\u0026uuml;ceer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and marked (*) in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition of goat cheeses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c10\" namest=\"c3\"\u003e \u003cp\u003eCheese\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHS2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e38.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e47.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e47.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e41.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e47.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e47.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e43.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e46.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e47.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eAab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eAab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e43.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eAd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e44.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eAd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e47.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e48.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFat/TS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e47.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e51.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e49.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e51.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e52.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" 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\u003cp\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. *Data (day 1) was adapted from Uzku\u0026ccedil; \u0026amp; Karag\u0026uuml;l Y\u0026uuml;ceer, (2023). Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking; TS, % total solids; Fat/TS, fat in total solids; Protein/TS, protein in total solids; Salt/M, salt in moisture. Statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eSimulated in vitro digestion\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e digestion of cheese samples was carried out according to a standardised static \u003cem\u003ein vitro\u003c/em\u003e digestion method suitable for food proposed by Minekus et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], using enzymes α-amylase from porcine pancreas (A1031), pepsin from porcine gastric mucosa (P7012), pancreatin from porcine pancreas 8xUSP (P7545) and porcine bile extract (B8631). After \u003cem\u003ein vitro\u003c/em\u003e digestion procedure, a centrifuge process was carried out at 9000 rpm and middle layers of centrifuged samples was used for determination of degree of hydrolysis and low molecular weight (\u0026lt;\u0026thinsp;3 kDa) fractions (LP). LP containing potential bioactive peptides in the samples were fractionated using Vivaspin Turbo 15 (Sartorius, Gloucestershire, United Kingdom) centrifugal filter units with a cut-off of 3 kDa (3000 MWCO) in accordance with the filter manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eAssessment of hydrolysis degree (DH)\u003c/h3\u003e\n\u003cp\u003eThe amino acid and peptide concentration (serine equivalent) of \u003cem\u003ein vitro\u003c/em\u003e digested samples was used as an indication of protein hydrolysis in the end the pancreatic phases of the digestion and measured as described by Nissen et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. DH data were corrected with the data obtained in the control digestion.\u003c/p\u003e\n\u003ch3\u003eDetermination of ACE-I activity\u003c/h3\u003e\n\u003cp\u003eACE-I activity was determined by the method of Cushman and Cheung [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Accordingly, 20 \u0026micro;L LP of \u003cem\u003ein vitro\u003c/em\u003e digested cheese was added to 100 \u0026micro;L 5 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e hippuryl-histidy-leucine (HHL, Sigma) substrate solution (dissolved in 0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sodium borate buffer solution containing 0.3 M NaCl, pH 8.3). After adding 40 \u0026micro;L of 0.1 U/mL ACE (from rabbit lung, Sigma) reagent to this suspension, the reaction mixture was further incubated at 37 \u0026ordm;C for 30 min and finished with 150 \u0026micro;L of 1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HCl. Then 800 \u0026micro;L of the organic phase was transferred to a glass tube and then dried out by heating at 95\u0026deg;C for 20 min to evaporate ethyl acetate. The residue hippuric acid was dissolved in 1 mL H\u003csub\u003e2\u003c/sub\u003eO and absorbance was measured at a wavelength of 228 nm (Shimadzu, UV-1800 UV\u0026ndash;vis, Japan). The result was expressed as ACE-I activity %, calculated using the equation: (A\u0026thinsp;\u0026minus;\u0026thinsp;B)/(A\u0026thinsp;\u0026minus;\u0026thinsp;C) \u0026times; 100, where A is the absorbance without sample, B is the absorbance without ACE, and C is absorbance in the presence of both ACE and sample.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant capacity of the LP extracted from \u003cem\u003ein vitro\u003c/em\u003e digested cheese was determined according to ABTS radical scavenging and CUPRAC methods. Trolox equivalent antioxidant capacity (TEAC) of the cheeses according to the ABTS decolorization assay was performed by mixing 10, 20, and 30 \u0026micro;L of samples and 1000 \u0026micro;L of ABTS solution in quartz micro cuvette, respectively. After 6 min incubation period, the absorbance of the solution was measured at 734 nm using a spectrophotometer (Shimadzu, UV-1800 UV\u0026ndash;vis, Japan). TEAC values were reported as mM of Trolox equivalents per 100 mg cheese [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For CUPRAC method; 70 \u0026micro;L copper (II) solution (10 mM), 70 \u0026micro;L neocuproin solution (7.5 mM) and 70 \u0026micro;L ammonium acetate buffer (1 M) were added into the microplate wells, respectively, and 7 \u0026micro;L sample was added followed by 70 \u0026micro;L distilled water. The microplate was shaken well for 10 s, sealed and kept at room conditions for 30 min. At the end of 30 min, absorbance was measured at a wavelength of 450 nm (NanoQuant Infinite M200, Tecan, Switzerland) against the blank. Antioxidant activities were reported as mM Trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCheeses were compared using general linear model ANOVA at each ripening day to determine effects of cheesemaking process (heating process, starter culture addition and coagulant origin), and one-way ANOVA to evaluate effect of ripening time for each cheese. Comparison of means was carried out by using Tukey\u0026rsquo;s or Welch (for the non-parametric data) test. Data were obtained from repeated analysis and evaluated by Minitab 18.1 (Minitab 2017) software. Results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error to indicate the precision of the sample mean and account for variability in the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemical composition of cheeses\u003c/h2\u003e \u003cp\u003eTotal solids, fat/TS, protein/TS and salt/M of the cheeses are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. TS of cheeses were ranged between 38.56\u0026ndash;51.69%. The interaction of heat treatment and starter culture affected the TS of the cheeses (P\u0026thinsp;\u0026le;\u0026thinsp;0.01); however, use of plant or animal origin coagulating enzyme did not significantly affect (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01). TS ​​of the cheeses increased significantly during ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01), except HS cheeses, and the values changed approximately 7% in raw milk cheeses and 12% in HF cheeses.\u003c/p\u003e \u003cp\u003eTotal solid of cheese is directly affected by the composition of milk, cheese production technique and ripening condition. The salt moves towards the center of the cheese mass and disperses in the structure of the cheese during salting process. The dispersion of salt continues throughout ripening until the salt is completely homogeneously distributed in the cheese mass. In a study reporting similar results to this study; Uzku\u0026ccedil; et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] reported that there was no difference between the TS ​​of goat cheeses produced from milk coagulated with \u003cem\u003eC. cardunculus\u003c/em\u003e protease or animal rennet, and TS ​​increased during ripening. The changes in TS ​​during 90 days ripening were attributed to salt migration, which led to whey or water release from the cheese[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The fact that RF, RS and HF cheeses ripened for 90 days have higher TS than fresh cheeses can be attributed to similar reason. Similar increase in TS result also observed in Serbian traditional goat cheese during ripening [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The fact that the TS ​​of HS1 and HS2 cheeses did not change during the ripening period can be explained by referring to the pH, titratable acidity results presented on the previous publication of the cheeses in this study. Accordingly, this may be due to the fact that HS cheeses reached their final pH (4.52) and titratable acidity (1.96) values ​​on the 1st day of ripening and the rest of the ripening period did not show the ripening process characteristics observed in the other cheese groups [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The role of pH in cheese texture is particularly important because changes in pH are related directly to chemical changes in the protein network of the cheese [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Barac et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] found significantly increased TS during 50 days of ripening in traditional Serbian goat cheeses produced by heat treatment at 90\u0026deg; C for 10 min, ranging from 44.88\u0026ndash;49.45%, which are comparable to the cheeses produced from heat-treated milk in the present study. However, Kocak et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] reported significant increase in moisture (decrease in TS) content of goat cheese ripened in brine during storage, attributing the change to the movement of water from the brine to the cheese curd as a consequence of the low salt concentration in the brine (\u0026lt;\u0026thinsp;20%). Unlike the results, in this study, no decrease in dry matter was observed because the cheeses were not stored in brine during the ripening period. The TS results determined in this research during ripening were also comparable to the TS reported for goat milk cheeses in other studies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fat/TS ratios of the cheeses were ranged from 47.70 to 56.89% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Heat treatment, starter culture addition, plant coagulant or their interaction did not affect significantly the fat/TS of the cheeses (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01). Fat/TS of HF cheeses increased at the end of the ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01), this change may be attributed to a high rate of TS change in ripened cheeses compared to the fresh cheeses. In fresh soft goat cheeses produced from pasteurized milk using thermophilic and mesophilic starter cultures, fat/TS ratios were found between 49.8 and 54.2% during 60 days ripening [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], similar to the fat/TS finding in this study. The fat/TS [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and protein/TS contents of the different goat cheeses are generally comparable with the result in the present study, although there are minor differences due to the composition of raw milk and cheese-making procedures.\u003c/p\u003e \u003cp\u003eThe effect of interaction between heat treatment and starter culture addition on the protein/TS results of the cheeses was significant only 60th and 90th days of ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). The protein/TS ratios of cheeses ranged between 45.14 and 49.33% on the 1st day of ripening, while the values were determined between 38.20 and 42.42% on the 90th day of ripening. Popovic-Vranjes et al.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] reported fat/TS ratios between 38.42 and 44.78% in goat cheeses, which were similar with the present study. The protein/TS ratios of cheeses decreased during ripening in all cheeses (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Protein/TS findings in the present study were similar to the findings presented by Miloradovic et al.[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] for high heat-treated fresh goat (Saanen) cheeses (46.87\u0026ndash;48.85%). However, lower protein/TS ratios than the present study were reported by Barac et al.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] for white brined goat cheese and by \u0026Aacute;lvarez and Frenso[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] for all age Palmero cheese (26.98\u0026ndash;31.37%). The higher protein/TS results than different goat cheeses [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] may be attributed to the milk source (Palmero Goat) and cheesemaking techniques (different salting and ripening methods).\u003c/p\u003e \u003cp\u003eThe salt/M ratio of the cheeses were ranged between 4.76\u0026ndash;6.54% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The interaction effect of heat treatment and starter culture addition on the salt/M of the cheeses for day 1 was significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). HF cheeses (5.77\u0026ndash;6.54%) differed from the HS cheeses (5.13\u0026ndash;5.40) in terms of salt/M on the 1st day of ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). The salt/M of the cheeses did not change significantly during ripening (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A study on goat milk cheeses, which produced combination of dry (3%) and brine (8%) salting indicated salt/M contents from 1.76 to 5.67% during 40 days of ripening [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Salt/M findings in the present study is similar for 30\u0026ndash;40 days ripened cheeses, but different for fresh cheeses found by Miloradovic et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which can be attributed to the difference in salting technique and salting time. In addition, Kocak et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] also found similar salt/M ratios with this study for brined goat cheeses on ripening days but reported significantly increasing salt/M results during ripening. They expounded that the increase in salt/M content was expected during ripening due to salt diffusion from brine to cheese increases [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, statistically insignificant salt/M changes during ripening in this study may be attributed to the fact that cheeses were not ripened in brine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHydrolysis degree (DH)\u003c/h2\u003e \u003cp\u003eProteolysis is the breakdown of large and complex proteins into smaller and simpler peptides. In cheese, the presence of bioactive peptides is the result of a sensitive equilibrium between their release and their degradation by the activity of lactic acid bacteria proteinases and peptidases during cheese ripening [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Proteolysis results in the cheeses during cheese production and ripening processes were presented in our previous study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Accordingly, it was reported in detail that the water-soluble nitrogen in total nitrogen (WSN/TN) and total free amino acids (TFAA) results, which are some indicator analyses for proteolysis, were significantly affected by the interaction of heat treatment and starter culture addition. Moreover, WSN/TN and TFAA underwent noticeable increases during the ripening process of all cheeses. It was reported that HF ​​cheeses had limited proteolysis according to WSN/TN and TFAA, while HS group cheeses reaching values 30.26 (% in TN) and 12.72 (mg leu 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the end of the ripening. In addition, RP-HPLC peptide profiles were observed in the water-soluble fractions of the cheeses and Urea-PAGE analyses were observed in the water-insoluble nitrogen fractions, and the results were in accordance with the WSN/TN and TFAA results [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzymatic hydrolysis improves the functional properties of dietary protein by converting it into bioactive peptides while preserving its nutritional value. The digestion process has the noteworthy potential that proteolytic digestive enzymes can release bioactive peptides from goat milk proteins [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The hydrolysis of goat cheese protein and peptides with the \u003cem\u003ein vitro\u003c/em\u003e digestion was evaluated consulting the OPA assay based on the measurement in free NH\u003csub\u003e2\u003c/sub\u003e groups. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the resulting DH, total of salivary, peptic and pancreatic digestion, relative to the control condition. Heat treatment, starter culture addition, plant coagulant or their interaction did not change significantly the DH of the cheeses (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01). After 2 min salivary, 2 h peptic and 2 h pancreatic of digestion, the average DH of cheeses was 86,43%. HF2 cheese had the lowest DH (81.38%) and HS2 cheese had the highest DH value (91.09%). This result can be attributed to the relatively low moisture content and/or higher salt concentration of HF2 compared to HS2. Higher moisture content can lead to greater salt solubility, and the salt concentration in cheese affects protein digestibility. Increased salt levels may decrease protein solubility [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], which can make protein digestion more difficult. The degree of hydrolysis fluctuated during ripening for in cheeses and no general trend was found for the release of free NH\u003csub\u003e2\u003c/sub\u003e groups by \u003cem\u003ein vitro\u003c/em\u003e digestion in cheeses on different ripening days. However, DH did not change significantly during ripening (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01). In a study examining the amount of peptide released from Ras cheese through \u003cem\u003ein vitro\u003c/em\u003e digestion, it was reported that, similar to our study, there was no difference between cheeses ripened for different periods [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eACE-I activity\u003c/h2\u003e \u003cp\u003eACE-I activity of LPs extracted from \u003cem\u003ein vitro\u003c/em\u003e digested cheeses that contains the potential bioactive peptides consisting of 2\u0026ndash;20 amino acids in cheese were presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The combination of heat treatment and starter culture addition significantly affected the ACE-I activity of the cheeses on all ripening days (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). Cheeses were divided into four groups according to their ACE-I activities on the 1st day of ripening. Initially, HF cheeses had the lowest (13.15\u0026ndash;15.52%) and HS cheeses had the highest (76.65\u0026ndash;77.94%) ACE-I activity ​​(Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). HF cheeses also had lower ACE-I activities ​​than other cheeses on 1st, 30th and 60th days of ripening while reaching similar ACE-I activity ​​as RS and HS cheeses on 90th day. 30, 60 and 90 days ripened RF cheeses had higher ACE-I activity ​​than the cheeses produced from heat-treated milk. However, it was found that cheeses with starter culture added had similar ACE-I activity ​​on the 30th, 60th and 90th days of ripening, regardless of the heat treatment and coagulant origin. The microflora of cheeses can affect the formation of bioactive peptides [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. It has been reported that raw milk cheeses show higher ACE inhibitor activity than cheeses produced from pasteurized milk, depending on the proteolytic enzymes of microorganisms in the natural microflora of milk [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In addition, starter cultures break down the ACE-I peptides in cheeses into peptides and amino acids with lower or higher activity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and \u003cem\u003eLactobacillus helveticus\u003c/em\u003e is generally more effective than \u003cem\u003eLactococcus lactis\u003c/em\u003e in the release of ACE-I peptides in cheese [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Saremnezhad et al. [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] reported the high ACE-I effect of the Lighvan cheese made from 100% raw goat's milk compared the cow milk cheeses, and they attributed to the amino acid sequence of the peptide fragments from goat's milk and the proteolytic power of the native microbiota of this type of milk. It has been reported that \u003cem\u003ein vitro\u003c/em\u003e digestion reduces the ACE-I activity in the water-soluble fractions of the cheeses, and this may be attributed to the loss of activity of ACE-I peptides as a result of hydrolysis by digestive enzymes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Ripening time had significantly affected ACE-I activity of cheeses (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). Increases and decreases were observed in the ACE-I activity of raw milk cheeses during ripening, and the highest ACE-I activity in cheeses were determined on the 30th and 60th days of ripening in RF cheeses (average values were 76.17 and 68.52%, respectively) and on the 30th days of ripening in RS cheeses (average value was 68.72%). In a study, the highest ACE-I activities ​​were determined in 60 days-ripened brined goat cheese produced using starter culture as 76.15% and 51.95%, respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Researchers reported increases and decreases in the ACE-I activity of cheeses during ripening [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Kocak et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] determined a decrease in the ACE-I activity of goat cheeses after the 60th day of ripening and attributed the decrease to the breakdown of active peptides into inactive form due to proteolysis. The average ACE-I activity of HF cheeses increased from 14.34\u0026ndash;47.59% during ripening, however, average ACE-I activity of HS cheeses decreased from 77.30\u0026ndash;44.56% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u0026Ouml;zcan Yardım and Durak [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] reported higher \u003cem\u003ein vitro\u003c/em\u003e ACE-I activity in goat cheese on 90th ripening day compared to sheep, cow and Van herby cheese. The lower ACE-I activity found in their goat cheese (10.44%) compared to all findings in our study may be due to differences in their digestion procedure for the water-soluble extract of cheeses.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eACE-I activities of goat cheeses (%)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eCheese\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRS2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHS2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37\u003csup\u003eBd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003csup\u003eBd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e76.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.39\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e77.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.23\u0026thinsp;\u0026plusmn;\u0026thinsp;5.48\u003csup\u003eAab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.21\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20\u003csup\u003eAab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e60.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.81\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.99\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e47.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.92\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.30\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.57\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003csup\u003eABab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eABab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45.61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e45.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e43.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eResearchers reported that the ACE-I activity of cheeses affected by milk pretreatment, starter cultures, scalding conditions, and ripening [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Torres-Llanez et al. [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] reported that ACE inhibitor peptides in unripened cheeses were formed by the proteolytic effect of starter cultures. Thus, the starter culture used in the production of HS cheeses provided the formation of peptides with high ACE-I activity in the cheese on the 1st day of ripening. The relatively low ACE-I activity determined in the first 60 days of ripening in the HF cheeses may be attributed to the lower proteolysis level in the absence of starter culture, thus lower bioactive peptide formation, which may be associated with the limited proteolytic activity of the residual milk-clotting enzyme. In addition, increase in ACE-I activity of HF cheeses on the 90th day of ripening might be attributed to the presence of lactococci and lactobacilli in those cheeses as a result of contamination starter or heat tolerant raw milk microflora explained in the previous study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity results of the LPs determined by the ABTS radical scavenging method are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The effects of heat treatment and starter culture addition on the antioxidant activities of cheeses on the 1st and 30th days of ripening was found to be significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). On the 1st day of ripening, RF1 and RF2 cheeses had the highest antioxidant activity (69.02\u0026ndash;65.46 mM Trolox 100 mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese), while HF1 and HF2 cheeses had the lowest antioxidant activity (45.95\u0026ndash;46.75 mM Trolox 100 mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese). In the cheeses ripened for 30 days, raw milk cheeses (RF and RS) had higher antioxidant activity than cheeses produced from heat-treated milk. The antioxidant activities of starter free cheeses (RF1-RF2 and HF1-HF2) changed significantly during ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). Saremnezhad et al. [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] reported the highest ABTS inhibition activity (61.26%) in the 90-day ripened Lighvan cheese made from 100% raw goat's milk, and they attributed the activity to structure of goat milk protein-forming amino acids and the proteolytic power of the enzymes present in the natural microflora of the milk. Antioxidant peptides are mostly released from caseins under the influence of starter and non-starter lactic acid bacteria [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Related to this statement, it was determined that natural microflora contributed to antioxidant activities in cheeses produced from raw milk among the cheeses stored for 30 days, and in addition, it was found that antioxidant capacities were higher among cheeses produced from heat-treated milk on the 1st and 30th days of ripening, depending on the starter culture activity added into HS1 and HS2 cheese milk.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eABTS antioxidant activities of goat cheeses (mM Trolox 100 mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eCheese\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRS2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHS2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eCc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e61.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003eBa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003eBCc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003csup\u003eBc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e59.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e54.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e58.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003eAB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e59.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.64\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e57.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCUPRAC results of LPs during ripening were presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The interaction of heat treatment and starter culture addition on the antioxidant activities of the cheeses in terms of CUPRAC was found to be significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01). The lowest antioxidant activities were determined as 9.01\u0026ndash;9.65 on the 1st day of ripening and 9.15\u0026ndash;9.41 mM trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese on the 30th day of ripening in HF cheeses, while the highest antioxidant activities ​​were determined as 16.88\u0026ndash;17.52 on the 1st day of ripening and 16.34\u0026ndash;15.88 mM trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese on the 30th day of ripening in RF cheeses, respectively. The antioxidant activities of raw milk cheeses decreased significantly during ripening (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) and the approximate average decrease rates were 21% in RF cheeses and 14% in RS cheeses. It can be interpreted that the enzymes originated from raw milk microflora released more low molecular weight peptides with antioxidant activity during the production of raw milk goat cheese. Accordingly, it was found that HF cheeses that were heat-treated but did not contain starter culture had lower antioxidant activity ​​than the other cheeses on the 1st, 30th and 60th days of ripening, and had similar results ​​with HS cheeses at the end of 90th day of ripening (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCUPRAC antioxidant activities of goat cheeses (mM Trolox g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cheese).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eCheese\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRS2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHS2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eABa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eBCa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eBCa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eABb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eCa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eCa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Abbreviations: R, raw milk; H, heat-treated milk; F, starter culture free; S, starter culture added; 1, coagulated by plant-origin rennet; 2, coagulated by calf rennet, codes indicate process factor in cheesemaking. Statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) differences among the means were marked with different lowercase letters in each row and different uppercase letters during storage for each parameter.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA few research showed that peptide formation in cheese production increases the antioxidant capacity of fresh cheeses, and the same quality increases during the ripening process [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Revilla et al. [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] found that the highest antioxidant activity (ABTS) in goat cheeses was determined on the 90th day of ripening, that producing cheese from milk obtained in different seasons differentiates the antioxidant activity, especially in fresh cheeses, and that summer cheeses have lower hydrophobic:hydrophilic ratios than winter cheeses, which results in strong proteolysis. Accordingly, they reported that fresh cheeses produced in summer months had higher antioxidant capacity. In a study investigating the antioxidant activities of white cheeses produced from cow and goat milk without adding starter culture, the antioxidant capacities of the water-soluble fractions of the goat cheeses, before and after \u003cem\u003ein vitro\u003c/em\u003e digestion, were 159.97 and 152.41 mM Trolox kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and it was found to be 25.33 and 81.43 mM Trolox kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the insoluble fractions, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In that study, it was stated that the antioxidant activities of goat cheese protein fractions increased significantly with \u003cem\u003ein vitro\u003c/em\u003e digestion, and the TEAC ​​determined on the 50th day of ripening were higher than the results ​​of less stored cheeses. It was reported that \u003cem\u003ein vitro\u003c/em\u003e digestion enhanced the antioxidant activity of 2-month-old fresh Kradi cheeses [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a study investigating the \u003cem\u003ein vitro\u003c/em\u003e antioxidant activities (ABTS and FRAP) of water-soluble fractions (\u0026lt;\u0026thinsp;3 kDa) of ripened cow, goat and Ezine cheeses produced by the same method, Turan and Durak [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] reported that goat cheese had a higher antioxidant capacity than other cheeses. In addition, the antioxidant activity differences between cheeses are related to the types of milk used in the production of cheese types as well as the number of peptides formed by the hydrolysis of the proteins of these milks to form different peptides during the proteolysis that occurs during the ripening of the cheeses. \u0026Ouml;zt\u0026uuml;rk and Akın [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] found that Tulum Goat cheese, produced by fermenting goat milk with its natural microflora and without applying heat treatment to the milk, had a higher antioxidant activity than cow Tulum cheese produced under the same conditions, according to the DPPH method, and this was due to the higher hydrophilic peptide content in goat milk Tulum cheese.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the bioaccessibility was assessed in the low molecular weight peptides of \u003cem\u003ein vitro\u003c/em\u003e digested the goat cheeses. Cheesemaking factors investigated in the study, heat treatment, starter culture addition and ripening, affected the bioaccessibility of goat cheese regardless of the source of milk coagulation enzyme used in production. Therefore, the goat cheeses showed ACE-I and antioxidant activities. It has been determined that in cheeses produced from heat-treated milk with the presence of starter culture, ACE-I activity decrease during ripening due to proteolytic activity of starter culture, but on the contrary, results increased in the absence of starter culture.\u003c/p\u003e \u003cp\u003eThe antioxidant activity was tested with two different assays ABTS and CUPRAC. For both tests, the free radical scavenging activity changed positively or remained unchanged following \u003cem\u003ein vitro\u003c/em\u003e digestion. The interaction of heat treatment and starter culture addition was affected the ABTS antioxidant activities of LP after \u003cem\u003ein vitro\u003c/em\u003e digestion of cheeses on the 1st and 30th days of ripening. ABTS values of RF group decreased, but the antioxidant properties of HF group increased during ripening while the antioxidant capacities of cheeses produced from starter culture added milk did not change. It was found that the CUPRAC values ​​of raw milk cheeses decreased with ripening, but the values ​​of heat-treated milk cheeses did not change.\u003c/p\u003e \u003cp\u003eIt was concluded that cheeses produced using plant-derived and animal coagulants were generally similar in terms of bioaccessibility. Accordingly, it has been shown that wild artichoke (\u003cem\u003eC. cardunculus\u003c/em\u003e) protease can be used as an alternative to produce goat cheese to obtain similar ACE-I and antioxidant activities. The findings of this work confirm that all age cheeses produced from raw milk without starter addition and fresh cheeses produced from heat-treated starter added milk, present over 50% ACE-I activity and can be a source of peptides with antihypertensive health-promoting properties. According to the results obtained in this study, raw goat milk cheeses have the potential to reduce oxidative damage by presenting bioactive peptides which have the antioxidant properties. To fully understand the health effects, it is necessary to conduct \u003cem\u003ein vivo\u003c/em\u003e studies. Further studies are required to put forward the new functional properties of goat cheeses and proteomic research to sequence peptides correlated to bioaccessibility properties and characterization bioactive peptides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is the part of PhD study of Hasan UZKUÇ under the supervision of Prof. Dr. Yonca KARAGÜL YÜCEER at the Department of Food Engineering, Faculty of Engineering, Çanakkale Onsekiz Mart University, Türkiye.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the Scientific Research Coordination Unit of Çanakkale Onsekiz Mart University with grant number: \u0026nbsp; FBA-2021-3585.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHasan UZKUÇ\u003c/strong\u003e: Conceptualization, writing – original draft, software, formal analysis, data curation, visualization. \u003cstrong\u003eYonca KARAGÜL YÜCEER\u003c/strong\u003e: Conceptualization, resources, writing – review and editing, supervision.\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethics requirements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human or animal subjects.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTUIK (2024) Turkish Statistical Institute- Livestock Statistics. 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LWT 63:837\u0026ndash;845. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2015.04.054\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2015.04.054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;zt\u0026uuml;rk Hİ, Akin N (2018) Comparison of some functionalities of water soluble peptides derived from Turkish cow and goat milk Tulum cheeses during ripening. Food Sci Technol (Brazil) 38:674\u0026ndash;682. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/1678-457x.11917\u003c/span\u003e\u003cspan address=\"10.1590/1678-457x.11917\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bioactive peptide, bioaccessibility, bioavailability, bioactivity, antihypertensive","lastPublishedDoi":"10.21203/rs.3.rs-6542067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6542067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to determine effects of heat treatment, starter culture addition, coagulant origin and ripening time on antioxidant and angiotensin-converting enzyme-inhibitory (ACE-I) activities of goat cheese. The bioaccessibility of the cheeses was tested in the low molecular weight (\u0026lt;\u0026thinsp;3 kDa) fractions (LP) of \u003cem\u003ein vitro\u003c/em\u003e digested samples containing bioactive peptides. The antioxidant activity of the cheeses was tested with ABTS [2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)] radical scavenging and CUPRAC (Cupric reducing antioxidant capacity) assays. The interaction of heat treatment and starter culture addition affected the bioaccessibility of the cheeses just as ripening process, while different coagulants had no effect. The highest ACE-I activities observed in heat-treated\u0026thinsp;+\u0026thinsp;starter-added milk cheeses on the 1st day and starter-free raw milk cheeses on 30th, 60th, 90th days of ripening. Starter-free raw milk cheeses exhibited better antioxidant activities in the ABTS assay on the 1st and 30th days, and CUPRAC assay throughout all ripening days.\u003c/p\u003e \u003cp\u003eObservations indicated that raw milk cheeses fermented by natural microbiota may exhibit an antihypertensive effect with over 50% ACE-I activity and reduce oxidative damage thanks to their bioactive peptides, demonstrating potential positive impacts on human health and suggesting their possible use in functional diets.\u003c/p\u003e","manuscriptTitle":"Effects of heat treatment, starter culture, plant coagulant and ripening on in vitro ACE inhibitory and antioxidant activity of goat cheese","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 19:33:44","doi":"10.21203/rs.3.rs-6542067/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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