Consuming raw milk has been associated with a reduction in risk of childhood asthma and atopy [1
] as well as respiratory infections [3
]. However, consumption of raw milk poses significant risks, due to potential presence of pathogens in raw milk [3
]. As an alternative to raw milk, specific milk ingredients for supplementing heat treated milk have become the focus of recent research, and a wide range of components have been hypothesized to be related to the allergy and asthma protective potential of raw milk versus commercially available milk [4
After industrial processing, cow’s milk considerably differs from raw milk in several aspects, with fat content and heat-treatment being the most obvious. Although the effects of fat content and heat treatment on reduction of asthma partially overlap, both factors exert strong independent effects [2
]. The effect of fat content was mainly attributed to the levels of ω-3 polyunsaturated fatty acids [2
]. Similarly, heat treatment reduced the levels of milk serum proteins such as β-lactoglobulin and α-lactalbumin, which in turn were found to be inversely related to asthma risk in children of the GABRIELA study with statistical significance [1
Although it remains open whether these proteins actually reduce the asthma risk themselves, these findings suggest an allergy preventive potential by heat-sensitive proteins in general. Generally whey proteins are susceptible to heat treatment [5
], particularly immunoactive proteins such as lactoferrin or lactadherin [7
]. Heating of heat-labile proteins results in denaturation and aggregation processes [8
] and thereby leads to a loss of biological functionality (e.g., lactoferrin [9
]). Denatured and aggregated proteins can be extracted from the milk with a combination of pH reduction and ultracentrifugation [10
], after which remaining levels of non-aggregated milk serum proteins can be determined [7
]. Besides denaturation and aggregation, heating may also lead to chemical modifications, especially the Maillard reaction [11
]. During industrial milk processing, relatively short heating times are applied, thus we expect relatively low levels of such chemical modifications, although especially for UHT processing a certain level of chemical modifications has previously been observed [12
]. The aim of this study was to assess the native protein profile of bovine milk serum after different industrially applied processing steps with varying heating intensity for the identification of potential asthma- and allergy-protective candidate proteins.
Heat treatment of milk led to a considerable decrease in number of detectable proteins and their levels of quantification with a clear relationship to the applied heat load. The most intensive treatment, i.e., boiling, reduced the number of proteins that could be detected by about 50% compared to raw milk, with the other heating types ranging in between. The various processing methods led to specific proteomic patterns covering 140 individual proteins as demonstrated by a cluster analysis. Of these, 23 distinct proteins were found to be substantially diminished in high heat treated milks. The majority of these heat-sensitive proteins were related to immune functions.
Typically, people in Westernized countries consume industrially processed milk and, increasingly, milk types with an extended shelf life. In addition, UHT milk with its very long storage duration of three months or more is nowadays very popular. Traditionally, commercially available milk had been pasteurized, i.e., heated at 72 °C for 20 s to inactivate potential hazardous microorganisms with only small gain in shelf life.
Despite the potential risk of life-threatening infections, a minority of people still consume raw cow’s milk, which has repeatedly been reported to protect against asthma, allergies, and respiratory infections in childhood [1
]. The wide consumption of cow’s milk thus renders it an attractive strategy for prevention if the risk of infections were to be overcome. An option might be the isolation and purification of the protective milk ingredients, and various studies have focused on the impact of industrial processing on the potentially beneficial molecules. At the same time, reducing heat load of commercially available dairy products may already lead to an increase in the availability of potentially immunoactive proteins.
Of the industrially applied processing steps, predominantly fat separation for adjusting milk fat levels, and homogenization for preventing fat creaming, affect the milk lipid fraction. However, homogenization also leads to a massive increase in fat globule surface, which will be covered by milk proteins, leading to a reduction of milk proteins in serum.
Waser at al., 2007 [23
] found an asthma and wheeze protective effect of milk fat containing products such as full cream milk and butter. In addition, Brick et al., 2016 [2
] implied the higher fat content and more precisely the higher content of anti-inflammatory omega-3 fatty acids in raw milk in the asthma protective effect of full cream milk obtained directly from a farm. Despite mild heating to 55 °C, high pressure treatment of milk (250 bar) used during the homogenization process has been found to profoundly rearrange protein quantity and structure [22
]. In addition, in the present study homogenization reduced total protein LFQ levels and specific protein detectability markedly.
Another major processing step is heating for destroying hazardous microorganisms and increasing shelf life. Thermo-labile milk components such as miRNAs [13
] and proteins may thus be involved in the protective effect of raw milk. Particularly protein functionality, solubility and quantity are all affected by intensity of heat treatment [7
]. Previously specific miRNA species were identified as possible contributors to the asthma-protective effect of farm milk [11
]. This notion is not in conflict with our current findings; rather both molecule classes might add to the effect or might even interact.
Loss et al., 2011 [1
] found inverse associations of asthma with higher levels of several milk whey proteins, i.e., bovine serum albumin, alpha-lactalbumin and beta-lactoglobulin. However, it remains unclear whether these specific proteins confer the effect themselves or whether they are proxies of heat labile proteins in general. Therefore, we quantified heat-induced alterations of the entire milk proteome by a comprehensive, standardized, and unbiased approach, i.e., without preselection of proteins.
First, we observed a considerable decrease of detectable proteins after heat treatment in a dose-dependent manner. Boiled cow’s milk contained the lowest number of detectable proteins, which is explained by the high heat load applied. The lower temperature of boiling compared to ESL or UHT is more than compensated by the much longer duration of the heating (Table 2
). In addition, the long heating time of boiling may also lead to more extensive chemical modification compared to industrial processes [11
], further reducing protein levels in these samples.
For further investigation in the impact of heating on the protein quantity and heat sensitivity, milk samples were categorized into high heat and no/low heat treated milk groups according to the clusters presented in the heat map (Figure 3
). This dichotomization was in line with findings on the first marginal transition of bovine whey proteins at about 81 °C [20
]. Actually, the difference between high and no/low heat treated samples was more than 25 °C with pasteurization not exceeding 72 °C and high heat treatment starting with 100 °C.
and Figure 4
describe some variance within milk types between the three farms, e.g., one of the UHT samples had a higher percentage of detectable proteins and a higher summed LFQ value than the respective other two UHT samples. Nevertheless, the heat map (Figure 3
) still groups all the high-heated samples together. Even though this UHT sample contains a higher overall protein intensity, and a higher number of identified proteins, the proteome profile still reflects a high heated sample. This might be due to a similar pattern in decrease of individual, heat sensitive proteins. The exact underlying mechanisms for these individual variations however cannot be explained in this study. Further investigations on a larger scale are needed to better understand the variability in proteome profile after heat processing.
The sum over all proteins, and more specifically the levels of 23 individual proteins were substantially lower in high heat treated samples as expected by previous work from Zhang et al., 2016 [7
]. Interestingly, most of these 23 particularly heat-sensitive proteins were related to immune functions (Table 3
), and several proteins have already been mentioned in the context of asthma and allergies. Under the assumption that some proteins withstand the acidity of the stomach milieu, they may be resorbed in the gut and exert physiologic functions. At least this has been suggested for e.g., lactoferrin (LTF) [27
], protease inhibitors [28
] and IgG [29
Among the most promising candidates was lactoferrin, which is known to stimulate the immune system by counteracting pathogenic invaders and injuries and preventing harmful overreactions of the immune system [30
Lactoperoxidase is a peroxidase enzyme secreted from the mammary gland that operates as a natural antibacterial agent [32
]. Asthmatic patients who were treated with lactoperoxidase aerosol showed lower disease activity and reduced damaging effects of hydrogen peroxide (H2
), which is mainly generated by neutrophils and eosinophils in asthma and contributes to airway damages and inflammation [33
Xanthine dehydrogenase/oxidase (XOR) might contribute to the formation of NO in the intestinal lumen and thereby exert antimicrobial properties [34
]. In our study we were unable to differentiate the rather similar variants, dehydrogenase and oxidase, as the only difference is an intramolecular change of two cysteines in the disulfide bond, whereas the amino acid chain, analyzed with the LC-MS/MS analysis, is identical.
In addition, a number of acute phase proteins such as fibrinogen, prothrombin, complement C3 and C9 were found to be highly heat-sensitive. How they may be involved in the anti-inflammatory effects ascribed to raw milk remains unclear, although the complement pathway, and specifically C3, has been implied in the development of allergy and asthma [35
Plakophilin-3 acts protective in both local and systemic inflammatory diseases [38
] and inter-alpha-trypsin inhibitor has anti-inflammatory, anti-scarring and anti-angiogenic properties [39
]. Protease inhibitors, including several inter alpha-trypsin inhibitors, have been found to be upregulated in the breast milk of allergic mothers and have been related to the pathogenesis of allergy and asthma [40
Lactadherin expression is found to be markedly reduced in asthmatic patients compared to healthy subjects, and suppresses airway smooth muscle hypercontractility [42
Polymeric immunoglobulin receptor may influence eosinophilic inflammation by binding secretory immunoglobulins [43
]. In addition, secretory components, which are part of the polymeric immunoglobulin receptor that can be cleaved off, have shown individual effects in mucosal immunity [44
Ultimately, the discovery of the CD14 molecule, a receptor of bacterial endotoxin, is interesting as gene–environment interactions of raw milk consumption and polymorphisms associated with this gene have been discussed controversially for childhood onset asthma [45
]. Similar to the human CD14 molecule, its bovine counterpart might transmit signals elicited by endotoxin, and thereby have an effect on the development or prevention of allergy and asthma.
Despite the plausible involvement of several proteins in the beneficial health effects we have to acknowledge that we cannot provide a direct link to disease status in this study. However, the palette of immune-active milk components detected in the present study can be seen as an extension to the findings by Loss et al., 2011 [1
], which explicitly linked protein levels to disease. In addition, this study only shows a decrease in native proteins, due to either denaturation or heat-induced chemical modification, without direct evidence for a loss-of-function. However, heating of milk has been shown to reduce biological activity of milk, including antibacterial capacity [6
] and previous studies showed a loss-of-function of milk immune proteins upon denaturation (e.g., Paulson, 1993 [47
]; Marin et al., 2003 [48
]). However, future studies are needed to investigate in the biological function of milk’s immunoactive proteins after applying heat treatments.
Another limitation of this analysis is the omission of the milk fat globule membrane (MFGM) fraction [49
]; their relatively low abundance in cow’s milk, however, precludes a major contribution to the effects by the entirety of immunoactive proteins present in milk. Our analyses were made after one freezing cycle; resulting alterations, however, seem to be very limited [7