Lowering the Maillard reaction products (MRPs) in heated whey protein products and their cytotoxicity in human cell models by whey protein hydrolysate

This study investigated the potential use of reconstituted whey protein hydrolysate as an antibrowning agent in thermally processed foods and as a chemopreventive ingredient in biological systems. Hydrolysates were prepared by tryptic (EC 3.4.21.4) hydrolysis of whey protein concentrate (WPC) or heated (80 °C for 30 min) whey protein concentrate (HWPC). Tryptic hydrolysis of WPC and HWPC increased the oxygen radical absorbance capacity-fluorescein (ORAC_FL) antioxidant capacity from 0.2 to 0.5 ??mol Trolox equivalent (TE)/mg protein in both whey protein hydrolysate (WPH) and heated whey protein hydrolysate (HWPH) (p < 0.05). The reconstituted WPH and HWPH could prevent the formation of Maillard reaction products (MRPs) induced by a thermal process employed on WPC suspensions between 80 and 121 °C in the presence of lactose up to 0.25 M (p < 0.05). The MRPs in HWPC were cytotoxic to both normal human intestinal FHs 74 Int cells and human epithelial colorectal carcinoma Caco-2 cells. The IC₅₀ of HWPC was around 3.18-3.38 mg/mL protein. Nonetheless, when both cell types were grown in media supplemented with WPH prior to the uptake of MRPs in HWPC at 3.5 mg/mL, they were able to survive (p < 0.05). Overall, this study indicated the efficacy of WPH and HWPH in the prevention of MRP cytotoxicity. It was suggested that the ORAC_FL antioxidant capacity of WPH and HWPH needed to be high enough to provide a chemopreventive effect against MRP cytotoxicity.     

Farm and factory production of goat cheese whey results in distinct chemical composition

To analyze differences in fat and protein content in cheese whey (CW) manufactured in cheese-making factories and farms, goat CW samples were obtained from 60 cheese-making farms and 20 cheese factories. Gross composition of samples was analyzed by using an MIRIS device (MIRIS Inc., Uppsala, Sweden), whey protein composition was subjected to electrophoretic analysis, and fatty acid composition was analyzed via gas chromatography. Goat CW from farms contained higher dry matter content (70.6 vs. 50.8 g/L, farms vs. cheese factories, respectively) and a higher fat percentage (10.5 vs. 1.2% over dry matter, farms vs. cheese factories, respectively) than CW from cheese factories. Analysis of individual proteins showed that CW from farms contained higher concentrations of lactoferrin (0.4 vs. 0.2 mg/mL of CW, farms vs. cheese factories, respectively) and caprine serum albumin (0.6 vs. 0.4 mg/mL of whey, farms vs. cheese factories, respectively) than CW from cheese factories. No differences were observed in the fatty acid profile. The main fatty acids present in goat CW were C16:0, C18:1, C14:0, and C18:0. Thus, the origin of CW affects gross composition and the protein profile, but not the fatty acid profile.     

Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk

Milk was fermented for up to 5 h at 43 °C with two lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus). A protease, flavourzyme, was added at the beginning of fermentation. The whey fraction was separated from the fermented milk and freeze-dried. During the 5 h of fermentation, the soluble protein content increased from 4.9 to 57.4 mg/g and peptide content increased from 2.1 to 32.8 mg/g, while inhibition of angiotensin I-converting enzyme (ACE) increased by a decrease of IC₅₀ from 0.708 to 0.266 mg/ml, respectively. The whey was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction of the whey showed the highest inhibitory efficiency ratio (IER) being 1329%/mg/ml. The amino acid sequence of the inhibitory peptide was Tyr-Pro-Tyr-Tyr, of which the IC₅₀ was 90.9 μM. The whey showed mixed-type inhibition kinetics, while Captopril, the positive control showed competitive inhibition on ACE. Their K_i values were 0.188 mg/ml and 0.0067 μg/ml, respectively. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced to 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml).     

Dopamine antagonist alters serum cortisol and prolactin secretion in lactating Holstein cows

The role of dopamine in regulating glucocorticoid and prolactin secretion was investigated in lactating Holstein cows by characterizing serum cortisol and prolactin responses to fluphenazine, a dopamine receptor antagonist. Twelve anovulatory cows received an intravenous bolus injection of either saline (n = 6) or 0.3 mg of fluphenazine/kg of body weight (n = 6) in wk 2 postpartum. Blood samples were collected every 30 min for 4 h before and 4 h after saline or fluphenazine injection. Serum progesterone concentration was 0.13 ± 0.1 ng/mL and did not differ between groups. No difference in serum cortisol concentrations was detected between groups before treatments. Fluphenazine increased serum cortisol concentrations within 30 min after fluphenazine administration (>30 ng/mL) and concentrations remained elevated throughout the sampling period. Cortisol remained unchanged in saline-treated cows (<10 ng/mL). Prolactin concentrations also increased after fluphenazine administration (103.1 ± 3.1 ng/mL), but were unaffected by saline (18 ± 3.1 ng/mL). Prolactin concentrations remained elevated throughout the sampling period in fluphenazine-treated cows. Our results indicated that a dopamine antagonist increased cortisol, suggesting that endogenous dopamine, at least in part, regulates cortisol and prolactin secretion. These effects are regulated through dopamine receptors in anovulatory lactating dairy cows during the early postpartum period.     

The effect of starter culture and annatto on the flavor and functionality of whey protein concentrate

The flavor of whey protein can carry over into ingredient applications and negatively influence consumer acceptance. Understanding sources of flavors in whey protein is crucial to minimize flavor. The objective of this study was to evaluate the effect of annatto color and starter culture on the flavor and functionality of whey protein concentrate (WPC). Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a mesophilic lactic starter culture or by addition of lactic acid and rennet (rennet set). Pasteurized fat-separated whey was then ultrafiltered and spray dried into WPC. The experiment was replicated 4 times. Flavor of liquid wheys and WPC were evaluated by sensory and instrumental volatile analyses. In addition to flavor evaluations on WPC, color analysis (Hunter Lab and norbixin extraction) and functionality tests (solubility and heat stability) also were performed. Both main effects (annatto, starter) and interactions were investigated. No differences in sensory properties or functionality were observed among WPC. Lipid oxidation compounds were higher in WPC manufactured from whey with starter culture compared with WPC from rennet-set whey. The WPC with annatto had higher concentrations of p-xylene, diacetyl, pentanal, and decanal compared with WPC without annatto. Interactions were observed between starter and annatto for hexanal, suggesting that annatto may have an antioxidant effect when present in whey made with starter culture. Results suggest that annatto has a no effect on whey protein flavor, but that the starter culture has a large influence on the oxidative stability of whey.     

Available lysine and fluorescence in heated milk proteins/dextrinomaltose or lactose solutions

In order to predict and compare the effects of dextrinomaltose and lactose on available lysine loss by the Maillard reaction, six model systems were prepared by mixing casein, laboratory whey protein or commercial whey protein with dextrinomaltose or lactose. The solutions were prepared at concentrations similar to those used in enteral and infant formula processing and were heated at 100, 120 or 140 °C for 0–30 min. The progress of the Maillard reaction in these model systems was followed by monitoring free fluorescence intermediary compounds. Model systems with lactose showed higher available lysine less than the model systems with dextrinomaltose; linear lysine losses were obtained between 0 and 20 min at 100 and 120 °C. At sterilization temperature and time (120 °C/10 min), lysine losses of milk proteins with dextrinomaltose as reducing sugar were 6.1% for casein, 4.1% for laboratory whey protein and 13.4% for commercial whey protein. Available lysine showed correlation with furosine in model systems prepared with lactose and casein or laboratory but not commercial whey protein at 100 and 120 °C. The initial fluorescence value obtained by mixing casein or laboratory whey protein with lactose or dextrinomaltose was low (between 3.8 and 5.7), whereas the value obtained when commercial whey proteins were used was close to 9. At 120 °C/10 min, there was only a small increase of fluorescence in casein and laboratory whey protein but a large increase in commercial whey protein (threefold the initial value). Fluorescence measurement is useful for finding the extent of the Maillard reaction in commercial whey protein (thermally damaged protein). An absolute value greater than 10 may indicate that products were prepared with thermally damaged proteins.     

Short communication: Development of a novel method for the extraction of norbixin from whey and its subsequent quantification via high performance liquid chromatography

Norbixin is the primary carotenoid in annatto coloring, which imparts the desired orange color in Cheddar cheese. However, a portion of the colorant remains in the cheese whey and is undesirable; therefore, a bleaching step is often applied. Restrictions exist for norbixin concentrations in products destined for infant formula. As such, evaluation of norbixin concentrations in whey and whey ingredients is desirable. Current extraction methods are laborious and require solvents that are banned in many countries. The objective of this study was to develop a fast and inexpensive norbixin extraction and quantitation technique using approved solvents with similar sensitivity to current established methods. Instead of solvent extraction and column purification, acetonitrile was added directly to fluid wheys, retentates, and rehydrated whey protein concentrates. An isocratic mobile phase [70% acetonitrile and 30% water with 0.1% (wt/vol) formic acid] was used and, to increase sensitivity, a large volume (50 μL) was injected onto the column. The column used was a C18 column with a particle size of 2.6 μm and column length of 10 cm. The column inner diameter was 4.6 mm and the pore size was 100 ?. All of the previously described conditions allowed the run time to be only 4 min. The sample was sent through a photodiode array detector and quantified at 482 nm. Norbixin was quantified using external standard curves. The developed method had a >90% norbixin recovery in both milk and whey (9.39 μg/L–2.35 mg/L). The limit of detection of norbixin in fluid whey was 2.7 μg/kg and the limit of quantitation was 3.5 μg/kg, both of which are significantly lower than in previously described methods. The extracts were stable over 30 min at 21°C and stable over 24 h at 4°C. Repeatability and precision of the method had relative standard deviations of less than 13%. The developed method provides time and cost savings for evaluation of norbixin concentration in whey and whey products.     

Use of chitosan for selective removal of beta-lactoglobulin from whey

A method is described for selective removal of undenatured β-lactoglobulin from cheese whey based on interactions between whey proteins and chitosan. Whey was previously clarified at pH 4.5 with addition of chitosan (25 mg/100 mL), and selective removal of β-lactoglobulin was studied in the pH interval 4.6 to 6.5. Addition of chitosan caused selective precipitation of β-lactoglobulin that increased with pH. The content of β-lactoglobulin in whey decreased as the amount of chitosan added was increased. At pH 6.2, addition of 1.9 to 3.0 mg/mL of chitosan led to complete removal of β-lactoglobulin, whereas at least 80% of the rest of whey proteins remained in solution. The production of cheese whey without β-lactoglobulin could help to expand the applications of dairy by-products in food processing, and to isolate hypoallergenic whey protein concentrates.     

Short communication: Addition of milk replacer to colostrum whey: effect on immunoglobulin G passive transfer in Majorera kids

Forty-two Majorera kids (21 males and 21 females) were assigned to 3 groups, a colostrum group (C), a colostrum whey group (CW), and a colostrum whey plus milk replacer group (CWMR). All kids were fed twice on the first day and received 4 g of IgG/kg of body weight. No differences were found in serum IgG among the different treatments. Kid serum IgG concentrations on d 2 were 14.57, 17.25, and 13.32 mg/mL in the C, CW, and CWMR group, respectively. Labor time per animal was higher in the C and CW treatments than in the CWMR group (24.2 ± 2.3, 20.9 ± 3.4, and 16.1 ± 1.5 min, respectively). This new management system may decrease labor costs during the colostrum feeding period.     

乳铁蛋白对幼年大鼠骨健康影响

目的:研究乳铁蛋白(Lactoferrin,LF)对幼年大鼠骨健康的作用。方法:将40 只4 周龄雄性SD大鼠随机分为对照组(0 mg/kg bw/d)、LF低剂量组(100 mg/kg bw/d)、LF中剂量组(500 mg/kg bw/d)和LF高剂量组(1000 mg/kg bw/d),连续喂养30 d后,测定幼年大鼠体重和脏体比变化、股骨骨密度(Bone mineral density,BMD)和骨微观结构变化、血清及股骨骨髓组织中抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRACP)、基质金属蛋白酶9(Matrix metalloproteinase 9,MMP-9)、组织蛋白酶K(Cathepsin,CTSK)、核因子κB受体激活因子配体(Receptor activator for nuclear factor-κB ligand,RANKL)和骨保护素(Osteocalcin,OPG)水平。结果:灌胃LF 30 d后,与对照组比较,LF剂量组幼年大鼠体重、脏体比增长趋势相近,差异不显著(P>0.05),LF中、高剂量组骨小梁数量(Trabecular bone number,Tb.N)、骨小梁厚度(Trabecular bone thickness,Tb.Th)、血清及股骨骨髓组织中OPG和OPG/RANKL水平显著增加(P<0.05),骨小梁分离度(Trabecular bone space,Tb.Sp)、TRACP、MMP-9、CTSK和RANKL显著降低(P<0.05)。结论:补充LF能够减少幼年大鼠骨吸收,增加骨形成和骨量,调节骨代谢平衡,促进骨健康。     

Gossypol disrupts embryo development in heifers

Our objectives were to determine the effects of dietary free gossypol (FG) intake on plasma and uterine gossypol concentrations and embryo development and viability before and after culture with gossypol. Fifty postpubertal Holstein heifers weighing (±SD) 406 ± 34.5 kg at 11.5 mo of age were blocked by age and body weight (BW) and randomly assigned to 1 of 3 isocaloric and isonitrogenous diets differing in their FG content: control (0 mg of FG/kg of BW), moderate (17.8 mg of FG/kg of BW), and high (36.8 mg of FG/kg of BW). Heifers were fed the diets for 70 d before superovulation and embryo collection. Superovulated heifers were flushed on d 5 after induction of ovulation, and early morulae were either stained, to determine the number and proportion of live and dead cells, or randomly assigned to an in vitro culture for 96 h in media containing either 0 or 10 μg/mL of gossypol acetic acid. Plasma and uterine gossypol concentrations increased with increasing gossypol intake. The number of low-quality embryos-ova was greater for the high than for the moderate and control diets. Embryos collected from the high diet had the least number of cells because of fewer live cells, and were smaller in diameter. Greater dietary gossypol reduced blastocyst development and extended the time to reach the blastocyst stage. Similarly, gossypol concentration at 10 μg/mL compromised in vitro development and increased the proportion of degenerated embryos at 96 h in culture. These findings provide in vivo and in vitro evidence that intake of 36.8 mg of FG/kg of BW per d and gossypol concentrations >7 μg/mL in plasma, in uterine flush, or in vitro compromise early embryo development, which might explain some of the negative effects of gossypol on the fertility of dairy cows.     

Optimization of stress medium enhance hydroxyl radical inhibition by water-soluble protein from germinated millet

A growth-promoting medium was developed to enhance the production of a hydroxyl radical inhibitory water-soluble protein from germinated millet. The single factor test indicated that H₂O₂ plays a key role in the inhibition activity. An optimal medium composition, consisted of H₂O₂, gibberellin, Protamex and Tween-40, was achieved using statistical experimental designs. A fractional factorial design was applied to determine the key factors that affected the hydroxyl radical scavenging activity, and a central composite experimental design and response surface methodology were used to optimize those factors, respectively. A second-order polynomial prediction equation was obtained, that could determine the effects of H₂O₂ and Protamex on the response. It was found that a low concentration of H₂O₂ and Protamex could enhance the inhibition of hydroxyl radicals. The optimal medium composition for growth-promoting was as follows: H₂O₂ 1.56 mL/100 mL, gibberellin 0.2595 mg/L, Protamex 0.25 mg/mL and Tween-40 0.7 mL/100 mL. With the optimal medium, the highest hydroxyl radical inhibition (58.16%) was achieved.     

卵黄高磷蛋白磷酸肽及其钙螯合物在双细胞共培养体系中对成骨细胞分化的促进作用

研究了卵黄高磷蛋白磷酸肽（Phosvitin Phosphopeptide,PPP）及其钙螯合物（PPP-Ca）在成骨细胞（MC3T3-E1）和破骨前体细胞（RAW264.7）共培养体系中对成骨细胞分化的调节作用。对细胞毒性、碱性磷酸酶（AKP）、抗酒石酸酸性磷酸酶（TRAP）的活性和TRAP染色进行分析;用RT-PCR技术进一步探究成骨细胞RANKL/OPG通路相关蛋白的mRNA表达情况。结果发现,PPP和PPP-Ca可以使MC3T3-E1体系中的AKP活性分别增加9.5%和12.7%;PPP和PPP-Ca的加入可以使MC3T3-E1体系中OPG基因mRNA的表达量从0.92分别提升至1.25和1.39、使RANKL基因mRNA的表达量从1.00分别提升至1.23和1.45。该研究结果表明,PPP和PPP-Ca可有效促进成骨细胞的分化。实验结果为进一步探索磷酸肽在多细胞模型体系中的作用提供了研究基础,同时为磷酸肽的功能活性开发提供理论依据。     

Assessing nutritional quality of milk-based sport supplements as determined by furosine

Milk proteins have a strong position in the sport nutrition markets, such as sport supplements for highly trained athletes, apart from bodybuilders. Furosine, a well-known index for the availability of lysine and subsequently of the extent of the Maillard reaction, was evaluated in different common ingredients used for formulation, as well in commercial sport supplements. Furosine content ranged from 2.8 to 1125.7 mg/100 g protein in commercial sport supplements being usually lower in samples containing mainly whey protein isolates or casein, as compared with whey protein concentrates. It is estimated that 0.1–36.7% of the lysine content is not available in this type of products. The use of high quality ingredients for the manufacture of sport supplements reveals important, since it could be the major source of protein intake of certain group of consumers in high or moderate training regime. Furosine is an appropriate indicator to estimate the nutritional quality of sport supplements. A reference value of 70 mg furosine/100 g protein content in dried sport supplements could be set up for controlling the quality of milk-based ingredients used in the formulation. Samples with higher levels are suspected of use of low quality milk-based ingredients or inappropriate storage conditions.     

Oxidative modification of amino acids in porcine myofibrillar protein isolates exposed to three oxidizing systems

Susceptibility of amino acids in myofibrillar protein isolate (MPI) exposed to three oxidizing matrixes commonly encountered in muscle foods was compared. MPI suspensions (20 mg protein/mL) in 15 mM piperazine-N,N bis(2-ethane sulphonic acid) buffer (pH 6.0) were oxidized with an iron-catalyzed oxidizing system (IOS, 0.01 mM FeCl₃, 0.1 mM ascorbic acid, 0.0–10.0 mM H₂O₂), a lipid-oxidizing system (LOS, 0.0–10.0 mM linoleic acid, 3750 units of lipoxidase/mL), or a metmyoglobin (MetMb) oxidizing system (MOS, 0.0–0.5 mM H₂O₂/MetMb) for 24 h at 4 °C. Changes were quantitatively analyzed by determining amino acids on a reverse-phase liquid chromatographic (LC) system. In IOS, the amount of cysteine, methionine and tyrosine decreased (P < 0.05) with increasing [H₂O₂]. In LOS, only cysteine and methionine were lowered at increasing linoleic acid concentrations. In MOS, the quantity of alanine, cysteine, glycine, histidine, leucine and lysine, as well as the total amount of amino acids were significantly reduced at high concentrations of MetMb/H₂O₂. The results suggest that under typical meat processing conditions, iron- and metmyoglobin-catalyzed reactions play a major role in the oxidation of amino acids in muscle proteins.     

Effects of dietary energy and protein density on plasma concentrations of leptin and metabolic hormones in dairy heifers

The hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined in dairy cattle; consequently, our hypothesis was that leptin may fulfill this role. Our objectives were to determine the effects of diets differing in energy and protein density on dry matter intake (DMI), growth traits [body weight (BW), body condition score (BCS), back-fat (BF) thickness], and temporal changes in plasma concentrations of leptin, insulin, growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, and nonesterified fatty acids (NEFA) in dairy heifers during the pre- and postpubertal periods. In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted average daily gain of 1.10 (high, H), 0.80 (medium, M), or 0.50 kg/d (low, L). Five heifers in each of the H and L groups were further studied during period 2, either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The data provide evidence that 1) DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r² = 0.396); 2) unlike the acute postprandial increase in plasma insulin as a result of increased nutrient density (H 1.42 ± 0.09, M 1.02 ± 0.09, L 0.68 ± 0.11 ng/mL), plasma leptin concentrations did not respond acutely with a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, leptin at puberty did not differ among treatment groups (H 5.63 ± 2.48, M 4.28 ± 0.55, L 4.12 ± 0.72 ng/mL) and there was no evidence of an abrupt transition in prepubertal plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly growing heifers, but are apparently essential for puberty in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin (H 0.59 ± 0.07, M 0.43 ± 0.09, L 0.30 ± 0.09 ng/mL), IGF-1 (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL), and glucose (H 81.35 ± 3.39, M 73.59 ± 2.34, L 68.25 ± 3.39 mg/dL) reflected nutrient density, whereas GH (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and NEFA (H 209.54 ± 50.83, L 234.93 ± 48.97 μM) were inversely related to the plane of nutrition. Collectively, these data suggest that plasma concentrations of leptin may play a role in long-term regulation of energy reserves and puberty in growing Holstein heifers.     

Insulin stimulates glucose uptake via a phosphatidylinositide 3-kinase-linked signaling pathway in bovine mammary epithelial cells

The aim of this study was to investigate the effects of insulin on glucose uptake in lactating bovine mammary epithelial cells (BMEC). Primary BMEC were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 and treated with different levels of insulin (0, 5, 50, and 500 ng/mL) for 48 h after a 24-h starvation without fetal calf serum. Compared with the control cells (0 ng of insulin/mL), cell proliferation was enhanced by insulin treatment at all tested levels. Insulin significantly increased glucose uptake at a concentration of 500 ng/mL. In addition, the protein synthesis inhibitor cycloheximide (0.5 mg/mL) counteracted the insulin-elevated glucose uptake, thereby suggesting that newly synthesized transporter protein might take part in the insulin-induced glucose uptake. Furthermore, pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, did not influence the insulin-induced glucose uptake, but LY294002, a specific inhibitor of phosphatidylinositide 3-kinase, significantly reduced the insulin-stimulated glucose uptake. These results indicated that insulin-induced glucose uptake in BMEC may involve the phosphatidylinositide 3-kinase- but not mitogen-activated protein kinase-mediated signaling pathways.     

Relationship between cortisol response to stress and behavior, immune profile, and production performance of dairy ewes

The existence of a relationship between cortisol levels, after an acute stress, and behavioral activities, immunological profile, and production performance in sheep was studied. An initial flock of 30 Comisana ewes was involved in the experiment, and each of the 30 ewes was individually subjected to an isolation test in a novel environment. Subsequently, from the initial flock, 2 groups of 8 Comisana ewes were each retrospectively selected, and the animals were divided, according to their cortisol concentration 10 min after the isolation test, into high cortisol (HC) ewes, having a peak of cortisol concentration >90 ng/mL (average: 119.3 ng/mL ± 11.8), and low cortisol (LC) ewes having a peak of cortisol concentration <80 ng/mL (average: 52.4 ± 11.8). During the isolation test, the behavior of each animal was video-recorded and behavioral activities were registered. Blood samples were collected before the isolation test, immediately after the test (10 min), and at 60, 120, 300 min, 24 h, and 48 h after the test to evaluate percentages of T-helper (CD4⁺) and T-cytotoxic (CD8⁺) cells, CD4⁺/CD8⁺ ratio, and IL-1β and IL-6 levels. The ewes were milked for 3 d after the isolation test to determine cortisol levels and IL-1β and IL-6 concentrations in whey. Milk yield was recorded at each milking, and milk samples were analyzed for pH, nutritional parameters, renneting properties, and somatic cell count. During the isolation test, HC ewes exhibited a shorter duration of movement and fewer bleats than LC ewes. The average plasma IL-1β concentration was higher in HC than in LC ewes. The average whey IL-1β and IL-6 concentrations were higher in whey from HC ewes than in LC ewes. A positive correlation emerged between plasma and whey IL-1β concentrations. The average CD4⁺/CD8⁺ ratio in blood was lower in HC than in LC ewes. Time from isolation affected the CD4⁺/CD8⁺ ratio: at 120 min, the CD4⁺/CD8⁺ ratio increased compared with that at 10 min after isolation and then decreased until 300 min after isolation. On average, ewes with low cortisol concentrations showed higher milk production and lower SCC than ewes with high cortisol concentrations. Results suggest that plasma cortisol concentration is connected to the behavioral response and immune competence of dairy ewes and cytokine concentrations. Both whey IL-1β and IL-6 can be considered reliable indicators of the magnitude of hypothalamic-pituitary-adrenal axis activation. The stress-induced changes in CD4⁺/CD8⁺ ratio are critical for controlling disease incidence and planning appropriate vaccination programs. High reactivity of the hypothalamic-pituitary-adrenal axis is also associated with a reduction in milk production and an increased predisposition to develop intramammary inflammatory processes.