A Simple and Fast Detection Method for Bovine Milk Residues in Foods: A 2‐Site Monoclonal Antibody Immunochromatography Assay

The ingredient declaration on food labels assumes paramount importance in the protection of food‐allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β‐lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β‐lactoglobulin and 49 food samples labeled as not containing milk were negative for β‐lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β‐lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β‐lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as “containing milk” even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β‐lactoglobulin in foods. Practical Application: The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.     

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins

BACKGROUND: The main whey proteins α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of α‐LA and β‐LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS‐PAGE electrophoresis. RESULTS: Fermentation by lactic acid bacteria could significantly reduce the antigenicity of α‐LA and β‐LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, α‐LA and β‐LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS‐PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. CONCLUSION: The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity. Copyright © 2010 Society of Chemical Industry     

Innovative Uses of Milk Protein Concentrates in Product Development

Milk protein concentrates (MPCs) are complete dairy proteins (containing both caseins and whey proteins) that are available in protein concentrations ranging from 42% to 85%. As the protein content of MPCs increases, the lactose levels decrease. MPCs are produced by ultrafiltration or by blending different dairy ingredients. Although ultrafiltration is the preferred method for producing MPCs, they also can be produced by precipitating the proteins out of milk or by dry‐blending the milk proteins with other milk components. MPCs are used for their nutritional and functional properties. For example, MPC is high in protein content and averages approximately 365 kcal/100 g. Higher‐protein MPCs provide protein enhancement and a clean dairy flavor without adding significant amounts of lactose to food and beverage formulations. MPCs also contribute valuable minerals, such as calcium, magnesium, and phosphorus, to formulations, which may reduce the need for additional sources of these minerals. MPCs are multifunctional ingredients and provide benefits, such as water binding, gelling, foaming, emulsification, and heat stability. This article will review the development of MPCs and milk protein isolates including their composition, production, development, functional benefits, and ongoing research. The nutritional and functional attributes of MPCs are discussed in some detail in relation to their application as ingredients in major food categories.     

Towards a Comprehensive Validation of ELISA Kits for Food Allergens. Case 2—Milk

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.     

Inhibitor‐Assisted High‐Pressure Inactivation of Bacteria in Skim Milk

The combined inactivation effects of high hydrostatic pressure (HHP) and antimicrobial compounds (potassium sorbate and ε‐polylysine [ε‐PL]) on 4 different bacterial strains present in skim milk and the effect of these treatments on milk quality were investigated in this study. HHP treatment at 500 MPa for 5 min reduced the populations of Escherichia coli, Salmonella enterica Typhimurium, Listeria monocytogenes, and Staphylococcus aureus from 6.5 log colony‐forming units (CFUs) or higher to less than 1 log CFU/mL. Compared to HHP alone, HHP with potassium or ε‐PL resulted in significantly higher reductions in the bacterial counts. After 5 min of treatment with HHP (500 MPa) and ε‐PL (2 mg/mL), no growth of E. coli, S. enterica Typhimurium, or L. monocytogenes in skim milk was observed during 15 d of refrigerated storage (4 ± 1 °C). Scanning electron microscopy analysis revealed that the synergistic treatments caused more serious damage to the bacterial cell walls. Quality assessments of the treated samples indicated that the combined treatments did not influence the color, the turbidity, the concentrations of –SH group of the proteins, or the in vitro digestion patterns of the milk. This study demonstrates that HHP with potassium or ε‐PL may be useful in the processing of milk or milk‐containing foods.     

Structures and functionalities of milk proteins∗

During the last decade, marked progress has been made in the study of the fine details of the structures of milk proteins such as caseins, β‐lactoglobulin, α‐lactalbumin, and lactotransferrin. Many of the functional properties of the individual milk proteins, as well as the milk protein products, may be described at the molecular level. This article is an attempt to thoroughly review the three‐dimensional structures of major milk proteins, and to correlate them with the functional aspects of these proteins as food ingredients.     

Terpene profiles in Cantal and Saint‐Nectaire‐type cheese made from raw or pasteurised milk

Terpene profiles in cheese can be considered a ‘terroir’ fingerprint as the information contained in it should enable the pastures on which the animals were fed to be recognised. Yet a certain elasticity of the signature must be taken into account when determining authentication strategies, since products acknowledged as containing a common signature may have undergone certain procedures, such as cheese making and milk pasteurisation, that could have potentially altered their terpene profiles. In this study, Cantal and Saint‐Nectaire‐type cheeses were made from both raw and pasteurised milk from the same herd of dairy cows that had been grazed on natural grassland. Cheeses from raw and pasteurised milk were made from the same milking on the same days. Cantal and Saint‐Nectaire‐type cheeses were made on 4 different days, alternatively over four weeks. The terpenes in the cheese fat were analysed by dynamic headspace/gas chromatography/mass spectrometry. A great diversity of monoterpenes, sesquiterpenes and oxygen‐containing derivatives were identified. The major terpenes identified in most cheeses were β‐caryophyllene, α‐ and β‐pinene and limonene. Milk pasteurisation did not induce changes in the terpene profile of the cheese. Significant differences (p < 0.001) were observed between Cantal and Saint‐Nectaire cheeses: α‐pinene, β‐myrcene and β‐phellandrene were, respectively, three, five and five times more abundant in Cantal cheese, while tricyclene, α‐phellandrene and geraniol were found exclusively in Cantal cheese. In contrast, unidentified sesquiterpenes with retention indices (KI) = 1342 and 1511, α‐cubebene, longifolene and γ‐elemene were more abundant or exclusively found in Saint‐Nectaire cheese. A significant relationship with the date of milking (p < 0.01) was observed for α‐pinene and tricyclene in Cantal, for β‐myrcene, δ‐3‐carene, p‐cymene and α‐terpinene in Saint‐Nectaire cheese. Copyright © 2005 Society of Chemical Industry     

Concentrations of Volatile 4‐Alkyl‐Branched Fatty Acids in Sheep and Goat Milk and Dairy Products

Goat and sheep milk and dairy products thereof are characterized by a strong and unique flavor. In this context, the volatile minor fatty acid 4‐ethyloctanoic acid plays a prominent role along with 4‐methyloctanoic acid when both are present in free form. Using a novel GC/MS method in the selected ion‐monitoring mode, previously developed for sheep subcutaneous adipose tissue, we were able to analyze the total concentrations of these flavor‐relevant minor fatty acids as methyl esters in goat and sheep milk as well as in their products. Differences between the concentrations and ratios of 4‐methyloctanoic acid and 4‐ethyloctanoic acid in goat milk (n = 4), goat cheese (n = 4), sheep milk (n = 2), and sheep cheese (n = 4) were observed. Goat milk and cheese resulted in higher concentrations for both fatty acids (190 to 480 μg/g milk fat) and smaller 4‐Me‐8:0 to 4‐Et‐8:0 ratios (1.4 to 2.7) compared to sheep milk and cheese (78 to 220 μg/g milk fat; 4‐Me‐8:0 to 4‐Et‐8:0 ratio: 15 to 42). In all samples, the concentration of 4‐Me‐8:0 exceeded the one of 4‐Et‐8:0. However, due to its lower flavor threshold value the contribution of 4‐Et‐8:0 to the flavor was generally >76%. The calculated flavor values were >1400 for goat milk and cheeses and >200 for sheep milk and cheeses. In goat milk and its products, only a proportion of <0.1% 4‐alkyl‐branched fatty acids present in free form in the goat milk and <0.5% in the sheep samples would be sufficient to generate the characteristic goaty flavor. Parameters that promote or prevent the release of 4‐Me‐8:0, and especially 4‐Et‐8:0, will be decisive for the flavor in the resulting dairy product.     

A novel bi‐enzymatic system for lactose conversion

The potential of β‐galactosidase (β‐GAL) and glucose isomerase (GLI) to enhance the sweetening power of lactose and to generate galactooligosaccharides (GOS) and lactulose was studied. UF‐permeates of skim milk, sweet whey, acid whey and lactose solutions were incubated with β‐GAL and GLI. Lactose hydrolysis (LH) was 96–99%, glucose isomerisation (GI) about 53%. On a scale from 0 to 5, the intensity of sweetness increased from 1 to 3. The bi‐enzymatic process was also applied to yoghurt manufacture. Because of the inhibiting effect of calcium on GLI, the incubation was performed in UF‐permeates of skim milk. LH in yoghurt was 94%, whereas GI was 36% on average. Whey syneresis in yoghurt decreased from 12% to 5%. The sweetness increased from 1.4 to 2.9 on average. Applying the bi‐enzymatic system to 40% lactose solutions led to synthesis of about 20% prebiotic GOS. Products, such as 6‐galactobiose, allolactose and 6‐galactosyllactose were identified. About 1% lactulose was generated. Lactulose‐derived oligosaccharides were not found.     

食品中酪蛋白过敏原检测方法的比较

目的 比较市场上销售的三种检测牛奶中酪蛋白ELISA试剂盒效果。方法 以脱脂奶粉、酪蛋白做作阳性添加成分, 对三个品牌的ELISA试剂盒从回收率、精密度、特异性等方面进行比较。结果 ELISA试剂盒可以对多种食品进行酪蛋白含量检测, 回收率、精密度等各项检测性能指标均可满足相关检测低限要求。结论 ELISA检测试剂盒无需大型分析仪器、操作简便快捷、适用于大量样品快速初筛和各种基层实验室食品品质监控。     

Properties of casein concentrates containing various levels of beta‐casein

Microfiltration (MF) of milk was used to produce casein (CN) concentrates (80% protein) with reduced whey protein levels. By varying temperature of MF, we altered the proportion of β‐CN to αs‐CN in CN concentrates and compared them to milk protein concentrate (MPC). Casein content as a % of protein was approximately 90% for CN concentrates and approximately 80% for MPC. Smaller micelles and weaker rennet gels were observed for CN concentrates with low β‐CN level. Foam stability and yield stress values were higher for CN concentrates with a high β‐CN level. Modified CN concentrates can be produced by altering the proportions of individual CNs.     

Iodide Residues in Milk Vary between Iodine‐Based Teat Disinfectants

Majority of iodine found in dairy milk comes from the diet and teat disinfection products used during milking process. The objective of this study was to evaluate the effects of 4 iodine‐based teat dips on milk iodide concentrations varying in iodine level (0.25% vs. 0.5%, w/w), normal low viscosity dip versus barrier dip, and application method (dip vs. spray) to ensure safe iodine levels in dairy milk when these products are used. The iodine exposure study was performed during a 2‐wk period. The trial farm was purged of all iodine‐based disinfection products for 21 d during a prestudy “washout period,” which resulted in baseline milk iodide range of 145 to 182 ppb. During the experiment, iodine‐based teat dips were used as post‐milking teat disinfectants and compared to a non‐iodine control disinfectant. Milk iodide residue levels for each treatment was evaluated from composited group samples. Introduction of different iodine‐based teat disinfectants increased iodide residue content in milk relative to the control by between 8 and 29 μg/L when averaged across the full trial period. However, residues levels for any treatment remained well below the consumable limit of 500 μg/L. The 0.5% iodine disinfectant increased milk iodide levels by 20 μg/L more compared to the 0.25% iodine. Compared to dip‐cup application, spray application significantly increased milk iodide residue by 21 μg/L and utilized approximately 23% more teat dip. This carefully controlled study demonstrated an increase in milk iodide concentrations from iodine disinfectants, but increases were small and within acceptable limits.     

The effects of refined barley β‐glucan on the physico‐structural properties of low‐fat dairy products: curd yield,microstructure, texture and rheology

The beneficial role of dietary fibre in human nutrition has lead to a growing demand for incorporation of novel fibres, particularly barley β‐glucans, into foods. Barley β‐glucans are regarded as dietary fibre ingredients that are partially soluble in water. The aim of the present work was to investigate the possibility of using barley β‐glucan in milk systems in relation to the coagulation properties of milk containing β‐glucan, and to the rheology, texture and microstructure of fresh curds. The rate of coagulation and optimum coagulum cutting time were evaluated using rheological measurements. Results show that coagulation/gelation time of the milk can be reduced significantly with the incorporation of β‐glucan; curd yield increased and the viscoelastic properties of the curd were altered with β‐glucan additions. The relationships between curd rheological behaviour and its microstructure are discussed in relation to use of novel hydrocolloids in dairy processing. The results suggest that barley β‐glucan has the potential to be used as a fat replacer in low‐fat dairy systems. Copyright © 2004 Society of Chemical Industry     

Concentration characteristics of bovine β‐defensin 1 and 2 in fresh bovine milk and infant formula

The aim of this study was to determine the concentration of defensins BNBD‐1 and BNBD‐2 in fresh bovine milk and infant formula, and to compare the defensin concentrations of bovine milk and human milk. Abundant BNBD‐1 and BNBD‐2 was found in fresh bovine colostrum. However, compared to fresh bovine milk, BNBD‐1 and BNBD‐2 concentrations in infant formula were very low. The BNBDs concentration ranges were also less than β‐defensins in human milk. The BNBD‐2 concentration in fresh bovine milk was higher than that of BNBD‐1.     

Development of a method employing reversed‐phase thin‐layer chromatography for establishing milk fat purity with respect to adulteration with vegetable oils

A systematic study was conducted using reversed‐phase thin‐layer chromatography wherein unsaponifiable matter of samples namely, pure cow and buffalo milk fats, vegetable oils (groundnut oil, soya bean oil and sunflower oil) and milk fats adulterated with vegetable oils (≥1%) were run along with the reference standards using two new solvent systems to detect adulteration in milk fat. The results of the study revealed that adulteration at even a 1% level could easily be detected using this rapid, reliable and reproducible method based on the presence of β‐sitosterol as a marker and some additional spots ascribable to their occurrence in vegetable oils only.     

Effect of digestive enzymes on the activity of camel‐milk insulin

This study investigated the effect of digestive enzymes on the activity of camel‐milk insulin. The digestion was performed using the sequential action of pepsin and pancreatin. Proteolysis degree was estimated using the O‐phthaldialdehyde method. Insulin concentration was determined using an enzyme‐linked immunosorbent assay (ELISA). Results revealed that milk proteins were partially digested by pepsin alone and the degradation was increased during the pepsin–pancreatin digestion as compared to control. Insulin lost its activity after 30 min of pepsin digestion, and it was not detected by ELISA. This study strongly suggests that insulin is not responsible for the antidiabetic action of camel's milk.     

Membrane Fractionation Processes for Removing 90% to 95% of the Lactose and Sodium from Skim Milk and for Preparing Lactose and Sodium‐Reduced Skim Milk

ABSTRACT: Pilot‐scale microfiltration (MF), microfiltration‐diafiltration (MDF), ultrafiltration (UF), ultrafiltration‐diafiltration (UDF), and nanofilration (NF) membrane fractionation processes were designed and evaluated for removing 90% to 95% of the lactose and sodium from skim milk. The study was designed to evaluate several membrane fractionation schemes as a function of: (1) membrane types with and without diafiltration; (2) fractionation process temperatures ranging from 17 to 45 °C; (3) sources of commercial drinking water used as diafiltrant; and (4) final mass concentration ratios (MCR) ranging from about 2 to 5. MF and MDF membranes provided highest flux values, but were unsatisfactory because they failed to retain all of the whey proteins. UDF fractionation processes removed more than 90% to 95% of the lactose and sodium from skim milk. NF permeate prepared from UDF cumulative permeate contained sodium and other mineral concentrations that would make them unsuitable for use as a diafiltrant for UDF applications. A method was devised for preparing simulated milk permeate (SMP) formulated with calcium, magnesium, and potassium hydroxides, and phosphoric and citric acids for use as UDF diafiltrant or for preparing lactose and sodium reduced skim milk (L‐RSM). MF retentates with MCR values of 4.7 to 5.0 exhibited extremely poor frozen storage stabilities of less than 1 wk at ?20 °C, whereas MCR 1.77 to 2.95 MDF and UDF retentates and skim milk control exhibited frozen storage stabilities of more than 16 wk. L‐RSM exhibited a whiter appearance and a lower viscosity than skim milk, lacked natural milk flavor, and exhibited a metallic off‐flavor.     

Effect of pH on the formation of serum heat‐induced protein aggregates in heated yak milk

Serum heat‐induced protein aggregates at various pH levels were isolated from heated yak milk by size‐exclusion fast protein liquid chromatography. Analysis by reversed‐phase high‐performance liquid chromatography showed that β‐lactoglobulin and κ‐casein were the two main proteins in these aggregates. The amount of serum heat‐induced protein aggregates increased from pH 6.6 to 7.4, whereas the content of β‐lactoglobulin in aggregates decreased from 61.9% at pH 6.6 to 50.4% at pH 7.2, and then increased to 51.3% at pH 7.4. The content of κ‐casein, αs₁‐casein and β‐casein increased from pH 6.6 to pH 7.4. These results show that serum heat‐induced protein aggregates are pH dependent.     

Isolation,purification, and alteration of some functional groups of major milk proteins: A review

This review covers selected methods of isolation and purification of mainly α_s‐casein, β‐casein, κ‐casein, β‐lactoglobulin, and α‐lactalbumin. Selected methods of alteration of some functional groups of these proteins also were reviewed. Isolation and purification of milk proteins per se are methods of modifying the individual milk proteins. Gram quantities of these proteins can now be purified in a relatively short time using ion‐exchange resins. Due to the prominent use of non‐food‐grade reagents in the procedures for preparation of these milk proteins, individual proteins are not maximally utilized for the manufacture of food/feed and pharmaceutical products. Therefore, intensive research efforts are needed to obviate the problems associated with underutilization of milk proteins.