Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies

1 Scope

Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo.

2 Methods and results

Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin. Whey‐specific IgE levels were measured to determine sensitization and immune cell populations from spleen, mesenteric lymph nodes and Peyer's patches after oral whey administration were measured by flowcytometry. Whey‐specific IgE and IgG1 levels in partial whey hydrolysate sensitized animals were enhanced, but challenge did not induce clinical symptoms. This immunomodulatory effect of partial whey hydrolysate was associated with increased regulatory B and T cells in the spleen, together with a prevention of IgM‐IgA class switching in the mesenteric lymph nodes and an increased Th1 and activated Th17 in the Peyer's patches.

3 Conclusion

Partial hydrolysate sensitization did not induce whey‐induced clinical symptoms, even though sensitization was established. Increased regulatory cell populations in the systemic immune system and a prevention of increased total Th1 and activated Th17 in the intestinal immune organs could contribute to the suppression of allergic symptoms. This knowledge is important for a better understanding of the beneficial effects of hydrolysates.     

Evaluation of allergenicity of cow milk treated with enzymatic hydrolysis through a mouse model of allergy

Cow milk (CM) allergy is a worldwide concern. Currently, few studies have been performed on the immunoreactivity of CM and fewer still on the antigenicity of CM in vivo and in vitro. In this study, we assessed the potential allergenicity of enzymatically hydrolyzed CM using in vitro ELISA and oral sensitization and challenge of BALB/c mice. Alcalase-, Protamex-, and Flavourzyme-treated CM (all from Novozymes) diminished IgE binding capacity, with greatest reductions of 56.31%, 50.62%, and 56.45%, respectively. Allergic symptoms and levels of total IgG₁ were reduced, and allergic inflammation of the lung, jejunum, and spleen was relieved. Moreover, the numbers of CD8⁺ T and B220⁺ cells decreased, and the balance of CD4⁺ T/CD8⁺ T cells was effectively regulated. These findings suggest that the potential allergenicity of CM was reduced by enzymatic hydrolysis, and our research will lay a solid foundation for developing high-quality hypoallergenic CM products.     

乳蛋白活性肽功能性的研究

介绍了乳蛋白肽的来源及国内外发展概况，通过人体试验证明了乳蛋白肽的调节免疫和抗氧化两大生物功效，并就其发展前景进行了讨论。     

Corn oil and milk enhance the absorption of orally administered allyl isothiocyanate in rats

Allyl isothiocyanate, a chief component of mustard oil, exhibits anticancer effects in both cultured cancer cells and animal models. The accumulation of the N-acetylcysteine conjugate of allyl isothiocyanate, the final metabolite of allyl isothiocyanate, in urine was evaluated in rats that were orally coadministered allyl isothiocyanate with fluids (e.g., water, green tea, milk, and 10% ethanol) or corn oil. The N-acetylcysteine conjugate of allyl isothiocyanate content in urine when allyl isothiocyanate (2 or 4 μmol) was coadministered with corn oil or milk showed a greater increase (1.4 ± 0.22 or 2.7 ± 0.34 μmol or 1.2 ± 0.32 or 2.5 ± 0.36 μmol, 1.6- to 1.8-fold or 1.5-fold, respectively) than when allyl isothiocyanate (2 or 4 μmol) was coadministered with water (0.78 ± 0.10 or 1.7 ± 0.17 μmol). This result demonstrates that corn oil and milk enhance the absorption of allyl isothiocyanate in rats.     

牛乳蛋白过敏儿童中主要过敏原的研究

目的:研究儿童牛乳蛋白过敏中主要过敏原的种类和含量.方法:采用间接酶联免疫吸附法测定儿童血清中的Ig水平.结果:血清中抗ás-CN、BSA和a-LG的特异性IgG水平与CMPA有关.特异性IgG4检测结果显示,a-Lg和BSA为主要过敏原.特异性IgE的多元线性回归结果为ás-CN、CN、a-Lg都对CMPA有显著性贡献,ás-CN的标准回归系数最大(B=-0.589),a-Lg的最小(B=-0.271).结论:a-乳球蛋白(a-LG)、ás-酪蛋白(ás-CN)、和牛血清白蛋白(BSA)可能为黑龙江省牛乳蛋白过敏儿童的主要过敏原.     

An adjuvant-free mouse model to evaluate the allergenicity of milk whey protein

Milk allergy is the most common type of food allergy in humans with the potential for fatality. An adjuvant-free mouse model would be highly desirable as a preclinical research tool to develop novel hypoallergenic or nonallergenic milk products. Here we describe an adjuvant-free mouse model of milk allergy that uses transdermal sensitization followed by oral challenge with milk protein. Groups of BALB/c mice were exposed to milk whey protein via a transdermal route, without adjuvant. Systemic IgG1 and IgE antibody responses to transdermal exposure as well as systemic anaphylaxis and hypothermia response to oral protein challenge were studied. Transdermal exposure resulted in a time- and dose-dependent induction of significant IgE and IgG1 antibody responses. Furthermore, oral challenge of sensitized mice resulted in significant clinical symptoms of systemic anaphylaxis within 1 h and significant hypothermia at 30 min postchallenge. To study the underlying mechanism, we examined allergen-driven spleen cell T-helper 2 cytokine (IL-4) responses. There was a robust dose- and time-dependent activation of memory IL-4 responses in allergic mice but not in healthy control mice. These data demonstrate for the first time a novel transdermal sensitization followed by oral challenge mouse model of milk allergy that does not use adjuvant. It is expected that this model may be used not only to study mechanisms of milk allergy, but also to evaluate novel milk products for allergenic potential and aid in the production of hypo- or nonallergenic milk products.     

婴幼儿牛乳蛋白过敏症的研究进展

牛乳蛋白过敏症属于食品安全范畴,同时也涉及到临床医学领域.随着社会的发展,牛乳蛋白过敏越来越引起人们的关注.综述了婴幼儿牛乳蛋白过敏症的发生机制和分型、临床症状、检测手段、牛乳中引发婴幼儿牛乳蛋白过敏症的过敏原的改性等几个方面的研究进展.     

Effects of milk protein variants on the protein composition of bovine milk

The effects of β-lactoglobulin (β-LG), β-casein (β-CN), and κ-CN variants and β-κ-CN haplotypes on the relative concentrations of the major milk proteins α-lactalbumin (α-LA), β-LG, α_S1-CN, α_S2-CN, β-CN, and κ-CN and milk production traits were estimated in the milk of 1,912 Dutch Holstein-Friesian cows. We show that in the Dutch Holstein-Friesian population, the allele frequencies have changed in the past 16 years. In addition, genetic variants and casein haplotypes have a major impact on the protein composition of milk and explain a considerable part of the genetic variation in milk protein composition. The β-LG genotype was associated with the relative concentrations of β-LG (A » B) and of α-LA, α_S1-CN, α_S2-CN, β-CN, and κ-CN (B > A) but not with any milk production trait. The β-CN genotype was associated with the relative concentrations of β-CN and α_S2-CN (A² > A¹) and of α_S1-CN and κ-CN (A¹ > A²) and with protein yield (A² > A¹). The κ-CN genotype was associated with the relative concentrations of κ-CN (B > E > A), α_S2-CN (B > A), α-LA, and α_S1-CN (A > B) and with protein percentage (B > A). Comparing the effects of casein haplotypes with the effects of single casein variants can provide better insight into what really underlies the effect of a variant on protein composition. We conclude that selection for both the β-LG genotype B and the β-κ-CN haplotype A²B will result in cows that produce milk that is more suitable for cheese production.     

蛋白饲料源小肽对奶牛产奶量和乳品质的影响

在奶牛精饲料中补充添加实验室独立研发生产的蛋白饲料源小肽产品,验证小肽对泌乳期奶牛的增乳及其改善乳品质的作用.试验组随机分为3组(小肽分别按日粮的0.1%,0.3%,0.6%添加).结果表明:试验期各组奶牛产奶量比试验前显著升高(P＜0.01),分别提高2.0%,6.1%,7.4%;乳脂率、乳蛋白质量分数都有较大提高,乳脂率分别提高0.8%,2.1%,2.9%,乳蛋白分别提高1.4%,1.7%,2.0%:乳糖质量分数及干物质质量分数也都有明显提高;从实际生产经济效益分析,0.3%添加量组要好于其他两组.     

人乳与牛乳天然小分子蛋白肽的差异对比及功能分析

研究利用超滤技术和Tricine-SDS-PAGE电泳将人乳与牛乳中天然存在的小分子蛋白肽进行分离。蛋白条带经质谱鉴定发现,人乳小分子蛋白肽含有28种,牛乳小分子蛋白肽含有24种,二者存在18种相同蛋白肽。Gene Ontology(GO)功能注释分析发现,在生物过程中,人乳小分子蛋白肽发挥的作用要高于牛乳小分子蛋白肽,尤其体现在免疫过程中的作用;在分子功能上,二者主要分子功能是结合作用,其中人乳小分子蛋白肽的结合作用强,而牛乳小分子蛋白肽参与的转运活性的分子功能大于人乳小分子蛋白肽;在细胞组成上,二者均参与了细胞与细胞膜的组成。通过Kyoto Encyclopedia of Genes and Genomes(KEGG)代谢通路分析可知,人乳小分子蛋白肽参与酪氨酸和抗原的加工和呈递2个代谢通路,而牛乳小分子蛋白肽仅参与系统性红斑狼疮代谢通路。对人乳和牛乳中的小分子蛋白肽组成进行研究,不仅为揭示人乳蛋白和牛乳蛋白的差异及其生理功能提供了理论依据,而且为婴幼儿食品、母乳化制品达到母乳水平提供有益探索。     

Effects of enzymatic hydrolysis on the allergenicity of natural cow milk based on a BALB/c mouse model

Cow milk allergy is one of the most prevalent food allergies worldwide, particularly in infants and children. To the best of our knowledge, minimal research exists concerning the antigenicity of cow milk (CM). This study was performed to evaluate the allergenicity of enzymatically hydrolyzed cow milk (HM) in a BALB/c mouse model. The mice were randomly divided into 5 groups (n = 12/group), which were sensitized with phosphate-buffered saline, CM, and HM (Alcalase-, or Protamex-, or Flavorzyme-treated cow milk; Novo Nordisk; AT, PT, FT, respectively), respectively, using cholera toxin as adjuvant on d 0, 7, 14, 21. On d 28, the test mice were orally challenged with phosphate-buffered saline, CM, and HM (AT, PT, or FT) alone. Anaphylactic symptoms were monitored in the mice. Antibody, cytokine, histamine, and mouse mast cell protease-1 (mMCP-1) levels were measured using enzyme-linked immunosorbent assays. In addition, the numbers of T helper (Th)1 and Th2 cells, as well as the proportions of CD4⁺CD25⁺Foxp3⁺ Treg cells, in mouse spleens were detected using flow cytometry. Statistical significance was determined by one-way ANOVA. The results revealed significant differences between CM- and HM-challenged mice. Among these, the clinical scores of HM-challenged mice (AT, 1.50; PT, 2.00; FT, 1.92) were lower than those of CM-challenged mice (positive control, 2.83), but body weight and temperature of HM-challenged mice were higher than those of CM-challenged mice. In addition, significant reductions of allergen-specific IgE, IgG, histamine, and mMCP-1 were showed in HM-challenged mice, especially for histamine, ranging from 171.42 ng/mL to 214.94 ng/mL. Remarkable reductions of IL-4, IL-5, and IL-13 levels, as well as elevations of interferon-γ and IL-10 levels in the spleens of HM-challenged mice were also detected. Moreover, the number of Th2 cells decreased in the HM-challenged mice, to 2.36% (AT), 1.79% (PT), and 4.03% (FT), respectively, whereas the numbers of Th1 cells (AT, 6.30%; PT, 6.70%; FT, 6.56%) and the proportions of CD4⁺CD25⁺Foxp3⁺Tregs (AT, 8.86%; PT, 9.21%; FT, 9.16%) increased significantly. Our findings indicate that exposure to HM was sufficient to induce a shift toward a Th1 response, thereby reducing potential allergenicity. Importantly, these results will lay a theoretical foundation for the development of hypoallergenic CM products.     

Organochlorine pesticide residues in cow's milk from a tropical region of Mexico

A monitoring study was conducted to assess the magnitude of DDT [(1,1,1-trichloro-2,2-bis( p -chlorophenyl)ethane)] and HCH (hexachlorocyclohexane) contamination of bovine milk from the central tropical region of Mexico as the chemicals are extensively used in livestock and public-health programmes. Among pesticide residues analysed, the milk samples collected from Tlalixcoyan showed a mean level of γ-HCH (0.128 mg kg -1 ), which was significantly higher than residues in milk samples from Medellin (0.049 mg kg -1 ) and Paso San Juan (0.022 mg kg -1 ). The mean level of pp '-DDE in Medellin samples (0.039 mg kg -1 ) was significantly higher than in Paso San Juan (0.018 mg kg -1 ) and Tlalixcoyan (0.024 mg kg -1 ) milk samples. The pp '-DDT mean level from Medellin milk samples (0.089 mg kg -1 ) was significantly higher than the levels detected in the other two areas. The highest mean Σ-DDT level detected in Medellin samples (0.146 mg kg -1 ) was three times the FAO/WHO tolerance level. The highest acceptable daily intakes calculated for Σ-DDT were 0.017 μg kg -1 bw day -1 for adults and 0.530 μg kg -1 bw day -1 for infants; for γ-HCH residues, they were 0.021 μg kg -1 bw day -1 for adults and 0.666 μg kg -1 bw day -1 for infants, indicating that infants are more exposed to pesticide residues. Results indicate that cattle exposure to HCH and DDT results in high levels in dairy milk and a potential health risk for consumers.     

Effects of milk protein genetic variants on milk yield and composition

Effects of genetic variants of the milk proteins, alpha S1-casein, beta-casein, kappa-casein and beta-lactoglobulin (beta-lg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds. Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for beta-casein (B, A1B, A2 greater than A1A2) and beta-lg (B, AB greater than A) variants. Significant differences between beta-lg variants were also found with total solids (B, AB greater than A), casein (B, AB greater than A), whey protein (A greater than AB greater than B) and beta-lg (A greater than AB, AC greater than B greater than BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. alpha S1-Casein variants significantly affected alpha S1-casein (BC greater than B) and kappa-casein (B greater than BC) concentrations. beta-Casein variants affected concentration and proportion of beta-casein (A1B, A2B greater than A1, A1A2, A2, B), alpha S1-casein (A1, A2 greater than B) and kappa-casein (B greater than A2) and concentration of whey protein (A1 greater than most other beta-casein variants). kappa-Casein variants affected concentration and proportion of kappa-casein (B greater than AB greater than A), proportion of alpha S1-casein (A greater than AB greater than B) and concentrations of beta-lg (A greater than AB, B) and alpha-lactalbumin (A, AB greater than B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.     

乳蛋白ACEI酶解工艺条件的研究

通过测定牛乳蛋白酶解液的ACE抑制活性，确定了蛋白酶水解牛乳蛋白产生ACE抑制肽（ACEI）的工具酶及其最佳酶解工艺备件。结果表明：在胰蛋白酶，蛋白酶A，蛋白酶M，肽酶R四种酶中，蛋白酶A为酶解牛乳蛋白制备ACEI工具酶；其最佳酶解工艺条件为温度50℃，pH值为7．0，酶用量为3％，底物浓度为5％，酶解时间10h，在该条件下得到的酶解液1C50为0.8987g／L；在最佳酶解工艺条件下得到的酶解液经柱层析分离，在2个较大洗脱峰处得到的ACEI活性较高，其抑制活性分别为84．98％和85．35％，相对分子质量分别为1340u和439u。     

Comparative performance of a mixed strain starter in cow's milk, ewe's milk and mixtures of these milks

Lactococcus lactis ssp. lactis IPLA 947, L. lactis ssp. lactis biovar. diacetylactis IPLA 838 and Leuconostoc citreum IPLA 616 and designed for Afuega'l Pitu cheese manufacture from pasteurized milk was assayed in cow's and ewe's milk, and in mixtures containing 10% and 20% ewe's milk in cow's milk. The evolution of microbial counts, pH, titratable acidity, organic acids and volatile compounds production throughout the incubation period was determined. The use of ewe's milk as a culture medium increased the metabolic activity of the starter culture reflected in a higher lactose consumption, significantly higher acidity and some carbon source-derived organic acids and volatile compounds production, as well as in slightly higher starter strains growth, although the latter was not statistically significant. Thus, it is suggested that ewe's milk or mixtures of ewe's with cow's milk can be satisfactorily used in the manufacture of this traditional cheese. Received: 27 August 1999