Antioxidants in milk

Studies on whey and deproteinised milk have shown that the major contributor to the total antioxidant capacity of whole milk is the casein fraction and that albumin is the major contributor to the total antioxidant capacity of whey protein⁽ Reference Zulueta, Maurizi and Frigola ^{28 )}. Another study compared the trapping of free radicals in milk, milk serum (obtained by centrifugation) and whey (obtained by acidification of skimmed milk) after subjecting the samples to size exclusion chromatography⁽ Reference Clausen, Skibsted and Stagsted ^{29 )}. As shown previously⁽ Reference Chen, Lindmark-Mansson and Gorton ^{30 )}, caseins contributed the most to the antioxidant activities measured by 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ORAC assays (89 %). β-Lactoglobulin and α-lactalbumin contributed much less to the total antioxidant capacity, and in the low-molecular-weight fractions, urate and ascorbate were identified as antioxidants. This last result was confirmed by Cloetens et al. ⁽ Reference Cloetens, Panee and Akesson ^{4 )} who published a review of the antioxidant capacity of milk (breast, animal and infant milks) with a focus on measurement methods. The major antioxidant in the low-molecular-weight fractions was identified as uric acid, an important milk antioxidant, which is also in agreement with previous data⁽ Reference Østdal, Andersen and Nielsen ^{31 )}. Uric acid contributes 60 % of the human plasma antioxidant capacity (ferric-reducing ability of plasma (FRAP) assay)⁽ Reference Benzie and Strain ^{32 )}. In milk, uric acid is formed from the ruminal breakdown of microbial nucleotides⁽ Reference Giesecke, Ehrentreich and Stangassinger ^{33 ,} Reference Johnson, Harrison and Riley ^{34 )}. Uric acid has both reducing and free radical-scavenging properties, which protect milk compounds from oxidative damage, and its antioxidative effect in milk is similar to that of ascorbate⁽ Reference Østdal, Andersen and Nielsen ^{31 )}. Other experiments have shown that the antioxidant activity of casein is mediated by its constitutive amino acids, while the antioxidant activity in whey is largely dependent on denaturation or partial hydrolysis and dominated by the free thiol groups of β-lactoglobulin⁽ Reference Clausen, Skibsted and Stagsted ^{29 )}. Clausen et al.⁽ Reference Clausen, Skibsted and Stagsted ^{29 )} also concluded that the number of major contributors in milk to the free radical-scavenging activity (ABTS and ORAC assays) is relatively low.

The study of Cekic et al.⁽ Reference Cekic, Demir and Baskan ^{35 )} took a similar approach: the antioxidant capacity of milk was measured using two assays, cupric-reducing antioxidant capacity (CUPRAC) and ABTS, because only these assays can assess the contribution of protein to the observed total antioxidant capacity. Different types of milk were tested: organic milk; pasteurised milk; UHT-processed fatty milk (3·0 % milk fat), low-fat milk (1·5 %) and skimmed milk (0·1 %); and homogenised milk in commercial packaging. For each milk type, three samples were analysed: defatted whole milk, a solution of milk protein and whey. Comparing the total antioxidant capacities of skimmed milk, casein, β-lactoglobulin, α-lactalbumin, and different protein-milk mixtures confirmed that milk proteins are responsible for a large part of the total antioxidant capacity of milk⁽ Reference Cekic, Demir and Baskan ^{35 )}.

Otherwise, β-lactoglobulin-depleted milk has been shown to possess approximately 50 % less antioxidant activity than raw skimmed milk⁽ Reference Liu, Chen and Mao ^{36 )}. Therefore, since β-lactoglobulin is very sensitive to thermal denaturation, dairy products should not be overheated to potentially maintain their antioxidant potential⁽ Reference Liu, Chen and Mao ^{36 )}. Although β-lactoglobulin is generally considered a mild antioxidant, the authors hypothesised that ‘it is possible that the free thiol groups in β-lactoglobulin or in other milk proteins act as a reducing agent to regenerate other antioxidants in the raw milk’⁽ Reference Liu, Chen and Mao ^{36 )}. Concerning the mechanisms, Liu et al.⁽ Reference Liu, Chen and Mao ^{36 )} showed that β-lactoglobulin is a mild antioxidant (<vitamin E) but the conversion of β-lactoglobulin monomers into dimers is partly responsible for the in vitro LDL protection against oxidation induced by Cu. In addition, β-lactoglobulin substantially loses its antioxidant activity through crosslinking the thiol groups due to heating (100°C for 2 min) or through carboxymethylation to block the thiol groups, with Cys-121 playing an essential role.

Cervato et al.⁽ Reference Cervato, Cazzola and Cestaro ^{37 )} evaluated the antioxidant properties of milk casein subunits (α-, β- and κ-caseins) in liposome models. All casein subunits can inhibit the peroxidation of arachidonic acid inserted in multilamellar liposomes of dipalmitoylphosphatidylcholine, but α-casein appears to have the greatest effect. This observation could be attributed to the Fe-chelation ability of phosphoserine residues, for which ten, five and one are present in α-, β- and κ-casein, respectively. Caseins appear to favour self-oxidation of Fe and thus inhibit lipid peroxidation⁽ Reference Maubois and Leonil ^{38 )}. However, these authors believe that mechanisms other than simple Fe chelation are involved in the antioxidant activity of casein, indicating that caseins exhibit a variety of mechanisms in their inhibition of peroxidation, involving both the specific properties of other proteins and characteristics of caseins. Finally, milk proteins and digested peptides might contribute to protecting the endothelial cells of the digestive tract against oxidative injury by free radicals.

Power et al.⁽ Reference Power, Jakeman and FitzGerald ^{39 )} recently summarised laboratory results on antioxidant peptides, particularly those derived from milk, including their antioxidant potential measured in vitro and evaluated in vivo, mechanisms of action, bioavailability, potential applications and health benefits with a particular focus on ageing populations and regulatory requirements. One of the main important points is that these peptides, released during enzymic hydrolysis, are inactive when included in the ‘parent’ protein before digestion. The released peptides were shown to have the ability to scavenge free radicals, chelate metal ions, and/or inhibit lipid peroxidation. However, as explained by Power et al. ⁽ Reference Power, Jakeman and FitzGerald ^{39 )}, because of the variety of methods used to assess their antioxidant activity, data comparison from one study to another is difficult. Otherwise, the mechanisms of action of the antioxidant peptides derived from whey and casein have not been fully elucidated. Preliminary studies suggested that these mechanisms are related to the amino acid composition, sequence/structure and hydrophobicity, i.e. physico-chemical properties of the amino acids, of the peptides⁽ Reference Chen, Muramoto and Yamauchi ^{40 )}. In some cases, peptides derived from milk proteins have in vitro antioxidant activity similar to or even higher than that of chemical antioxidants (for example, butylated hydroxytoluene or butylated hydroxyanisole)⁽ Reference Power, Jakeman and FitzGerald ^{39 )}. Further, the authors gave an overview of the antioxidative peptides derived from cows’ and human milk protein following enzyme hydrolysis, including their amino acid sequences and the nature of the corresponding fragments. The authors concluded that such antioxidant peptides could find appropriate applications for specific populations such as the elderly because of the increased risk of sarcopenia and oxidative stress with ageing⁽ Reference Power, Jakeman and FitzGerald ^{39 )}.

The study by Woo et al.⁽ Reference Woo, Jhoo and Kim ^{41 )} confirmed that hydrolysis of milk proteins is effective at increasing their antioxidant activity (ABTS assay). Specifically, the tryptic digest of casein showed the highest radical-scavenging activity, and the casein hydrolysates that passed through a membrane with a threshold of 3 kDa exhibited the highest antioxidant activity. In addition, Şanlidere Aloğlu⁽ Reference Şanlidere Aloğlu ^{42 )} determined the antioxidant capacity of raw, pasteurised and sterilised milk before and after in vitro enzymic gastrointestinal digestion. The average values before digestion were 4·02, 4·47 and 4·18 mmol TE/g for raw, pasteurised and sterilised milk, respectively (ABTS assay), which significantly increased at the end of digestion to 11·13, 12·33 and 11·88 mmol TE/g, respectively. The main reason for this effect is that the antioxidant properties of casein and whey proteins were increased via hydrolysis in the presence of many digestive enzymes and that certain peptides released after the digestion of milk possessed an antioxidant capacity higher than that of other peptides⁽ Reference Hernández-Ledesma, Dávalos and Bartolomé ^{43 –} Reference Mandalari, Adel-Patient and Barkholt ^{45 )}. In this study, the reported values obtained via the ABTS assay were mostly higher than those previously reported by Cloetens et al.⁽ Reference Cloetens, Panee and Akesson ^{4 )} obtained via the ORAC assay, emphasising the importance of using similar assays for relevant comparisons. While the ORAC assay is based on H atom transfer mechanisms and measures the oxidative degradation of a fluorescent molecule after being mixed with free radical generators, the ABTS assay measures the electron- or H-donating properties of antioxidants, both hydrophilic and lipophilic, and is based on the conversion of ABTS to its radical cation, which is reactive towards most antioxidants. However, as most studies have been conducted in vitro, the extension to the real antioxidant potential of hydrolysed peptides in vivo will require appropriate studies in human volunteers.

If the protein fraction is the most powerful hydrophilic antioxidant in milk, the most active lipophilic antioxidant is CLA, which is uniquely found in milk, ruminant meat and eggs from chickens that have been fed CLA and is characterised by conjugated double bonds⁽ Reference Grażyna, Hanna and Adam ^{7 )}. In vitro, CLA was shown to inhibit maize oil lipid peroxidation induced by Cu⁽ Reference Badr El-Din and Omaye ^{46 )} and to quench the stable radical DPPH•⁽ Reference Fagali and Catala ^{47 )}. In rats, CLA also protects structural lipids against free radicals and reactive oxygen species⁽ Reference Palacios, Piergiacomi and Catala ^{48 ,} Reference Kim, Kim and Ahn ^{49 )}. According to Dhiman et al. ⁽ Reference Dhiman, Nam and Ure ^{50 )} the total CLA in dairy products and milk varies from 0·12 to 0·68 % of the total fat. Milk from organic farms has a higher CLA content than conventional milk due to the higher content of PUFA in cows’ diets in organic farming⁽ Reference Bergamo, Fedele and Iannibelli ^{51 ,} Reference Rehberger, Bisig and Eberhard ^{52 )}, and Ferreiro et al.⁽ Reference Ferreiro, Gayoso and Rodriguez-Otero ^{53 )} report differences of +9·5 to 14·9 % in favour of organic milks. Lipophilic milk antioxidants also include vitamins A, D₃ and E, but milk is not a very rich source of vitamins E or D₃ ⁽ Reference Grażyna, Hanna and Adam ^{7 )}.

In addition to a protein fraction, antioxidant peptides, uric acid, CLA and lipophilic vitamins, milk also contains bioactive secondary phenolic compounds – such as equol, a metabolite of daidzein – which are formed from plant phenolics by the bacterial flora of the cow intestine⁽ Reference Mustonen, Tuori and Saastamoinen ^{54 )}. Tsen et al.⁽ Reference Tsen, Siew and Lau ^{55 )} analysed five commercial whole milk, four commercial semi-skimmed milk and three commercial skimmed milk samples purchased from local supermarkets in Singapore and found that the antioxidant capacity and the ability to inhibit the formation of F₂-isoprostanes increased with the concentrations of phenolic compounds and equol. However, differences in the antioxidant potential were not as marked as the differences in the equol concentration, indicating that other compounds contributed to the antioxidant potential. In addition, the equol and total phenolic concentrations were positively correlated with the milk fat concentration. While fresh full-cream milk contained 47·0 ng/ml equol and 0·70 μg gallic acid/g total phenolic compounds, fresh non-fat milk contained only 1·3 ng/ml and 0·04 μg gallic acid/g, respectively. These compounds are present in higher concentrations in the lipid fraction than in the aqueous fraction of cows’ milk; thus, the milk fat fraction of non-homogenised milk contained significantly higher amounts of phenolic compounds (2·42 v. 0·20 μg gallic acid/g) and equol (2·27 v. 0·13 ng/ml; P<0·05) than the corresponding aqueous fraction, respectively. Therefore, skimming may potentially reduce the levels of the original equol and phenolic compounds that otherwise may depend on the feeding practice of the cow.

Influence of animal species, fat content, thermal treatments and storage

The antioxidant capacity of milk differs according to the animal source: values of 1·0, 1·7, 1·7, 2·1 and 2·8 μmol TE/g (ABTS assay) were reported for human, cows’, horse, buffalo and goats’ milks, respectively⁽ Reference Oner, Sanlidere-Aloglu and Dedebaş ^{56 )}. The authors concluded that goats’ milk contains the highest concentrations of free radical scavengers than the others without giving further explanation. However, there is no doubt that the difference in antioxidant capacity according to animal origin is mainly due to the different composition of antioxidant compounds and/or the interaction of these compounds with the matrix to attenuate (by counteracting ion oxidation processes) or increase this potential. A higher antioxidant potential of goats’ milk compared with cows’ and donkey milk was also calculated by Simos et al.⁽ Reference Simos, Metsios and Verginadis ^{57 )}. These authors explained that the high antioxidant capacity of goats’ milk can be attributed to its richness in antioxidants, as well as the particular combination of compounds or their greater bioavailability.

According to the CUPRAC and ABTS assays, the highest total antioxidant capacity has been observed for fatty milks⁽ Reference Cekic, Demir and Baskan ^{35 )}. Skimmed milk has a 21 % (CUPRAC) and 6 % (ABTS) lower total antioxidant capacity than fatty milk. According to the authors, since fat has no antioxidant activity, total antioxidant activity of milk fat can be considered to be due to antioxidants entrapped in fat during milk homogenisation before UHT sterilisation.

Further, in the study by Zulueta et al. ⁽ Reference Zulueta, Maurizi and Frigola ^{28 )}, the fat content of the milk was not correlated with the total antioxidant capacity in UHT milk, while the total antioxidant capacity of low-fat pasteurised milk was significantly lower (13624–13984 μmol TE/l; ORAC assay) than that of milk with a higher fat content (14124–14216 μmol TE/l). Chen et al.⁽ Reference Chen, Lindmark-Mansson and Gorton ^{30 )} also observed that the total antioxidant capacity (ABTS assay) in commercial cows’ milk (3 % fat) was significantly higher than in cows’ milk with 0·5–1·5 % fat and skimmed milk. Such results suggested that interference with lipids and the reactivity of the lipophilic antioxidants and the fat globule membrane proteins may influence the total antioxidant capacity.

In addition to animal species and skimming, thermal treatments may also influence the milk antioxidant potential. For example, changes in potential antioxidants and pro-oxidants in pasteurised and heated skimmed milk in an oil bath at 80, 90 and 120°C for up to 24 h were measured⁽ Reference Calligaris, Manzocco and Anese ^{58 )}. At each heating temperature, the milk samples showed an initial increase in the pro-oxidant activity until approximately 5 h of thermal treatment, followed by a decrease in this activity until 24 h; the decrease was associated with an increase in the milk antioxidant activity, especially at 90°C and 120°C. Such changes were explained by both the thermal degradation of natural antioxidants and the formation of novel oxidised molecules in the early stages of the Maillard reaction. Therefore, short heat treatments may be responsible for the depletion of the overall antioxidant properties of milk. However, severe heat treatments provide recovery and even a possible increase in the antioxidant properties of milk due to the formation of brown melanoidins (Maillard reaction products). These results emphasise the important role played by the time–temperature combination of heat treatments, which could explain the apparent discrepancy with other studies showing no significant effect of temperature on the antioxidant capacity of milk.

In the previously mentioned study by Şanlidere Aloğlu⁽ Reference Şanlidere Aloğlu ^{42 )}, the average values for raw, pasteurised (72–75°C for 15–30 s) and sterilised (121°C for 15 min) milks were 4·02, 4·47 and 4·18mmol TE/g (ABTS assay), respectively, with no significant difference between these values. In a study of Zulueta et al. ⁽ Reference Zulueta, Maurizi and Frigola ^{28 )} the total antioxidant capacity of commercial UHT milks (ORAC assay) varied from 12·0 (sd0·5) to 14·5 (sd0·4) μmol TE/ml, and the capacity of pasteurised commercial milk ranged from 13·6 (sd0·7) to 14·2 (sd1·1) μmol TE/ml (ORAC assay), with no overall significant difference between the UHT and pasteurised milks. Similarly, heating the milk to 63°C for 1 h did not affect the total antioxidant capacity⁽ Reference Chen, Lindmark-Mansson and Gorton ^{30 )}. Therefore, Maillard reaction products resulting from heating appear to compensate for the heat-induced loss of organic antioxidant compounds.

Contrary to human breast milk, the influence of storage time on the antioxidant capacity of cows’ milk has rarely been studied. The most significant study measured the antioxidant capacity (FRAP assay) of six raw cows’ milk samples collected directly from a dairy and immediately cooled to 4°C for up to 48 h (i.e. refrigerated storage)⁽ Reference Amamcharla and Metzger ^{59 )}. The FRAP values of the raw milk significantly decreased with refrigerated storage time, i.e. –27 % upon 48 h of storage. According to the authors, this reduction in the total antioxidant capacity of milk can be attributed to constant and undefined interactions involving many pro- and antioxidants, but further details were not given.

Conclusions

Whole milk contains both hydrophilic (i.e. proteins and peptides, lactoferrin and antioxidant enzymes) and lipophilic antioxidants (i.e. lipophilic vitamins and CLA), and its overall total antioxidant activity will notably depend on the fat content and time–temperature combination applied (for example, Maillard products) (Table 2).

Table 2 Summary of the in vitro antioxidant capacity of the main dairy products

CLA, conjugated linoleic acid; SOD, superoxide dismutase; TAC, total antioxidant capacity; UHT, ultra-high temperature; ↓, decrease; ↑, increase.

Antioxidant potential

The antioxidant properties of naturally fermented milk result from the presence of casein, whey proteins, peptides, amino acids, uric acid, vitamins C, E, D and A, β-carotene, and enzymic systems (SOD, catalase, and glutathione peroxidase), as well as the presence of CLA and coenzyme Q₁₀ and the antioxidant activity of the lactic acid bacteria or kefir culture⁽ Reference Zulueta, Maurizi and Frigola ^{28 ,} Reference Jiménez, Antonia Murcia and Parras ^{60 –} Reference Pihlanto ^{62 )}. Fermented milks may therefore exhibit very different antioxidant potentials according to the origin of the milk and to the nature of the bacterial strain. Among different plain fermented products marketed in Poland (i.e. twelve yoghurts, twelve kefirs, two buttermilks, two cultured milks and a Turkish drink yoghurt called ayran), yoghurt and kefir were shown to have the highest antioxidant activity: the mean FRAP values were 1·19 (yoghurts), 1·03 (kefirs), 0·66 (cultured milks), 0·48 (ayran) and 0·45 (buttermilks) μmol Fe²⁺/g⁽ Reference Najgebauer-Lejko and Sady ^{63 )}. The ability to scavenge DPPH radicals was 0·19mmol TE/kg for the yoghurts and 0·17 μmol TE/g for the kefirs, followed by the buttermilks, cultured milks and ayran, for which the mean antiradical power was 0·14, 0·11 and 0·09 μmol TE/g (DPPH assay), respectively. The authors attributed this effect to the fortification of the yoghurt and kefir products with milk powder and/or milk proteins. In addition to a high protein content, the presence of additional probiotic strains of Lactobacillus casei also positively affected the values obtained from the FRAP and DPPH assays in this study.

Influence of the mammal species

The study by Balakrishnan & Agrawal⁽ Reference Balakrishnan and Agrawal ^{64 )} included the preparation of a fermented milk (with Pediococcus pentosaceus from cheese) from various sources such as cows’, goats’ and camel milk. The scavenging activity of milk (DPPH assay, %) after 24 h of fermentation was the highest with goats’ milk (93 v. 54 % before fermentation), followed by camel milk (84 v. 64 % before fermentation) and cows’ milk (78 v. 39 % before fermentation), illustrating an important increase of up to +39 % upon fermentation. A higher antioxidant activity (Trolox equivalent antioxidant capacity-ABTS assay) was also observed with camel milk fermented with Lactobacillus rhamnosus PTCC 1637 compared with cows’ milk⁽ Reference Moslehishad, Ehsani and Salami ^{65 )}. This was attributed to a higher proline content in the primary structure of camel milk caseins.

Influence of casein polymorphism

Perna et al.⁽ Reference Perna, Intaglietta and Simonetti ^{66 )} evaluated the antioxidant activity (FRAP and ABTS assays) of a yoghurt made from milk characterised by different casein haplotypes (α_s1-, β- and κ-caseins) and fortified or not with chestnut and sulla honey. A haplotype is a group of alleles of different loci located on the same chromosome and usually transmitted together. The antioxidant activity of the control yoghurt was 204·43(sd57·81) μg ascorbic acid equivalents/ml based on the ABTS assay and 116·74(sd29·30) μg ascorbic acid equivalents/ml based on the FRAP assay. The high antioxidant capacity of the yoghurt was attributed to several parameters, including the presence of different bioactive peptides from the proteolysis of milk proteins by lactic acid bacteria, the fermentation and post-acidification during storage that produce organic acids, and the possible peptide aggregation that occurs during the enzymic hydrolysis of whey protein and casein. Haplotypes BB-A²A¹-AA and BB-A²A¹-AB had the highest values in both antioxidant assays compared with the control yoghurt, clearly showing that milk protein polymorphism influences the antioxidant activity of yoghurt.

Influence of fermentation and storage

When comparing a skimmed milk with the corresponding fermented milk, Jiménez et al.⁽ Reference Jiménez, Antonia Murcia and Parras ^{60 )} observed that the fermented milk (46 %) was less effective than the skimmed milk (65 %) at inhibiting lipid peroxidation. However, both products trapped approximately 85 % of free hydroxyl radicals (^•OH). Moreover, based on the ABTS assay (Trolox equivalent antioxidant capacity), fermented milk had a significantly lower total antioxidant capacity of approximately 48 % relative to the skimmed milk after 6 min of measurement but had a capacity of +62 % after 24 h of measurement. No explanation was proposed for this unexpected effect.

The proteolytic systems of lactic acid bacteria are the main way to generate bioactive peptides, including both free radical-scavenging antioxidant activity and angiotensin-converting enzyme-inhibiting activity, during fermentation of the milk⁽ Reference Gobbetti, Minervini and Rizzello ^{67 )}. Evaluating the antioxidant properties of a skimmed milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340 revealed that among four peptide fractions of 10–5, 5–3, 3–1 and <1·0 kDa, those in the range of 5–3 kDa exhibited the highest antioxidant activity (66 %, DPPH assay), which was not significantly different from that of the 3–1 kDa peptide fraction (64 %)⁽ Reference Qian, Xing and Cui ^{68 )}. The DPPH free radical-scavenging effect of the <1·0 kDa peptide fraction reached 55 %. Furthermore, the antioxidant activity was correlated with the molecular weight from 0 to 5 kDa. However, considering the mucus layer in the small intestine, which has a thickness of 100–150 μm, molecular filters with an exclusion molecular weight of 0·6–0·8 kDa were used, and due to the peptidase in the intestinal epithelial cells, the authors suggested that ‘the antioxidant activity of the peptide fraction less than 1·0 kDa might represent that of fermentation better in vivo ⁽ Reference Cao and Zhang ^{69 )}’⁽ Reference Qian, Xing and Cui ^{68 )}. Qian et al.⁽ Reference Qian, Xing and Cui ^{68 )} reported results that agreed with those of Peng et al.⁽ Reference Peng, Xiong and Kong ^{70 )} showing that the 0·1–2·8 kDa fraction from whey protein isolation had the strongest free radical-scavenging capacity.

In a commercial fermented milk, researchers tried to identify the peptides included in the most active (angiotensin-converting enzyme and antioxidant activities) protein fractions isolated by reverse-phase HPLC-MS⁽ Reference Hernández-Ledesma, Miralles and Amigo ^{71 )}. Among several potentially active peptides, the β-casein peptide KVLPVPQ may possess antioxidant activity. Of the isolated fractions, the highest antioxidant activity was found in a fraction where seven of the eight peptides identified contained at least one proline residue, and six of them had more than two proline residues. Therefore, according to the authors, ‘the high content of proline peptides could determine the antioxidant activity found in this fraction’⁽ Reference Hernández-Ledesma, Miralles and Amigo ^{71 )}.

In a study by Aloğlu & Oner⁽ Reference Aloğlu and Oner ^{72 )}, the physico-chemical and microbiological properties of commercial and traditional yoghurts were determined during 4 weeks of storage, as well as the proteolytic activity and antioxidant activities (DPPH and ABTS assays) of peptides derived from hydrolysis. The antioxidant activity of water-soluble extracts of traditional yoghurt was higher than that of commercial yoghurt, reflecting differences in the chemical and microbiological properties. The physico-chemical and microbiological properties of traditional yoghurts generally do not meet the commercial standards. During storage for up to 4 weeks (4°C), the average values increased from 7·7 to 8·7 mmol TE/g for the commercial yoghurts and from 10·1 to 13·2mmol/g for the traditional yoghurt (ABTS assay). The presence and position of the amino acids tryptophan, tyrosine and methionine in the peptides were believed to be responsible for the antioxidant activity. Finally, if the initial total antioxidant activity of the yoghurts was low, the activity of the peptides released from the HPLC fractions was 10- to 200-fold higher.

Concerning the influence of cold storage (4°C), an increased antioxidant activity of fermented cows’ and camel milks was observed for up to 21 d due to an increase in proteolytic activity, but the increase primarily occurred between 14 and 21 d⁽ Reference Moslehishad, Ehsani and Salami ^{65 )}. Thus, the increase in proteolysis during storage increases the release of antioxidant peptides even more than fermentation.

Influence of the probiotic strain

The influence of the strain of probiotic on the antioxidant potential of fermented milks has been studied much more than the previous parameters. Most of these studies were based on the release of antioxidant peptides by different probiotics.

Studies have coherently demonstrated a correlation between an increased degree of proteolysis and the antioxidant potential. In the most recent study, Solieri et al.⁽ Reference Solieri, Rutella and Tagliazucchi ^{73 )} isolated non-starter lactobacilli from different cheeses because of their proteolytic activity and their ability to produce fermented milk enriched with angiotensin-converting enzyme and antioxidant peptides. Thirty-four strains of Lactobacillus from Parmesan cheese and five strains from Pecorino Siciliano (or Canestrato) were screened for their ability to hydrolyse milk proteins, and two strains from Parmesan, Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, were selected to inoculate sterile cows’ milk. The milk fermented with Lactobacillus casei PRA205 had a higher radical-scavenging activity (1·2(sd0·0) mmol TE/ml) than the milk fermented with Lactobacillus rhamnosus (0·9(sd0·1) mmol TE/ml) (ABTS assay). In addition to this effect, the increases in proteolytic and radical-scavenging activities measured during the fermentation of milk for 120 h were clearly correlated, confirming the important role of peptides in the antioxidant activity of fermented milks. Ramesh et al.⁽ Reference Ramesh, Kumar and Singh ^{74 )} studied the influence of different lactobacilli strains (n 19) from ten different species on the antioxidant capacity of the whey fraction of skimmed milk. The radical-trapping antioxidant activity ranged from 0·12 to 0·49 mmol TE/l (ABTS assay), confirming the low antioxidant capacity of the whey fraction. In agreement with previous results, the antioxidant and proteolytic activities of seven strains (i.e. Lactobacillus delbrueckii ssp. bulgaricus NCDC08, Lactobacillus plantarum NCDC25, Lactobacillus casei ssp. casei NCDC17, Lactobacillus rhamnosus NCDC24, Lactobacillus paracasei ssp. paracasei NCDC63, Lactobacillus fermentum NCDC141 and Lactobacillus helveticus NCDC288) were positively correlated. After the hydrolysis of casein with cell-free extracts from these seven strains, a significant increase in antioxidant activity was observed for Lactobacillus rhamnosus NCDC24, Lactobacillus casei ssp. casei NCDC17 and Lactobacillus paracasei ssp. paracasei NCDC63. For these three strains, low-molecular-weight peptides (<3·0 kDa) represented 60–70 % of the total antioxidant activity. Virtanen et al.⁽ Reference Virtanen, Pihlanto and Akkanen ^{75 )} studied the production of antioxidant activity (ABTS assay and lipid peroxidation) during fermentation with commonly used lactic acid ferments. Sterilised skimmed milk (120°C for 20 min) was fermented with twenty-five different strains of lactic acid bacteria, and the six with the highest antioxidant activity were selected. The whey fraction was isolated from each fermented milk for measurement of its antioxidant capacity. In both assays, the strains Leuconostoc mesenteroides ssp. cremoris, Lactobacillus jensenii (American Type Culture Collection (ATCC) 25258) and Lactobacillus acidophilus (ATCC 4356) exhibited the highest antioxidant activity. The development of antioxidant activity was correlated with proteolysis for four strains. Additionally, the highest hydrophobic amino acid content and proportion of peptides in the molecular mass range of 4–20 kDa were found within fermentates with the highest antioxidant capacity, while the other fermentates mostly contained large polypeptides and compounds below 4 kDa. The development of antioxidant activity was therefore clearly specific for the strain. Interestingly, the authors concluded that ‘The development of radical scavengers was more connected to the simultaneous development of proteolysis whereas lipid peroxidation inhibitory activity was related to bacterial growth’⁽ Reference Virtanen, Pihlanto and Akkanen ^{75 )}. Similarly, Sah et al.⁽ Reference Sah, Vasijevic and McKechnie ^{76 )} evaluated the effect of the probiotics Lactobacillus acidophilus (ATCC 4356), Lactobacillus casei (ATCC 393) and Lactobacillus paracasei ssp. paracasei (ATCC BAA52) on the antioxidant and anti-mutagenic activity of peptides extracted from yoghurt. Again, the degree of proteolysis by these probiotics was highly correlated with the antioxidant and anti-mutagenic activities of peptides, and the released peptides showed high antioxidant activity in the DPPH assay. The yoghurt containing the three probiotic strains together had the highest degree of hydrolysis and the highest antioxidant activity, emphasising a potential synergistic effect.

Other studies have generally compared the respective antioxidant potential of adding different probiotic strains and emphasised the synergistic action of combined strains compared with only one strain. A sterilised milk (unspecified thermal conditions) with 3·2 % fat was fermented with different microbial cultures, including a symbiotic culture of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus and monocultures of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium bifidus ⁽ Reference Gjorgievski, Tomovska and Dimitrovska ^{77 )}. For all cultures, the antioxidant activity of the fermented products was increased compared with non-fermented milk. The highest antioxidant activity was observed with Lactobacillus acidophilus, which neutralised 55 % of the free radicals, and the lowest activity was observed with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus (45 %). The authors suggested that the increased antioxidant activity was not only due to released peptides but also to free amino acids, enzymes and various other compounds. Similarly, Lim⁽ Reference Lim ^{78 )} evaluated the antioxidant properties of yoghurt fermented with lactic acid bacteria from pickled cabbage. Yoghurt fermented with Lactobacillus acidophilus PC16 had the highest antioxidant activity (DPPH assay) compared with yoghurt fermented with Lactobacillus casei PC05. Conversely, the ability to scavenge the superoxide anion and the activity of the enzyme SOD were significantly higher in yoghurt fermented with Lactobacillus casei PC05 compared with yoghurt fermented with Lactobacillus acidophilus PC16. Upon cold storage, there was no significant difference in the antioxidant activities of these yoghurts. In the study by Aleksandrova et al. ⁽ Reference Aleksandrova, Chikov and Velikova ^{79 )} four strains of Lactobacillus delbrueckii were selected based on their high proteolytic activity. All of the strains belonged to the subspecies lactis and bulgaricus and were used in combination with a commercial strain for yoghurt LBB BY D4 (LB Bulgaricum plc) to produce a yoghurt-type dairy product (from skimmed milk). The in vivo antioxidant activity caused by the peptides obtained during the fermentation of milk proteins was analysed in living yeast cells. The main results showed that the antioxidant capacity towards reactive oxygen species in the yeast cells was higher with the combination of strains than with only the commercial strain and that the different strains exhibited different antioxidant capacities. Osuntoki & Korie⁽ Reference Osuntoki and Korie ^{80 )} isolated lactobacilli from five native Nigerian fermented foods to ferment whey fractions from skimmed milk and measure their antioxidant activity (DPPH assay and lipid peroxidation inhibition assay). All the whey fractions had antioxidant activity, and the five isolates of lactobacilli with the highest activity were selected (Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii). The whey fractions were then fermented for 24 h with the selected micro-organisms. The antioxidant activity increased during the 24 h in all cases, but differently according to the strain; however, with Lactobacillus delbrueckii, the activity increased less and plateaued at 4 h. The authors further concluded that the fermented milk may serve as a vehicle for probiotic lactobacilli from non-dairy sources. Finally, Parrella et al.⁽ Reference Parrella, Caterino and Cangiano ^{81 )} studied the antioxidant capacity (ORAC and FRAP assays) of nine different samples of fresh skimmed (derived directly from the udder of the cow), pasteurised and UHT milks before and after fermentation with several lactobacilli, individually, and in combination with the yeast Saccharomyces boulardii. As expected, all fermented milk samples had greater antioxidant activity than the unfermented milk, with the effect being significant only for fresh milk. The co-incubation of the lactobacilli with yeast led to greater antioxidant activity, especially for fresh milk.

Specific case of kefir

Kefir is a fermented milk drink prepared by inoculating cows’, goats’ or sheep’s milk with kefir grains. The kefir grains initiating the fermentation are a combination of lactic acid bacteria and yeasts in a matrix of proteins, lipids and sugars. This symbiotic culture forms ‘grains’ that resemble cauliflower. Lactobacillus species are always present in kefir grains. Kefir appeared to have higher antioxidant potential but reduced glutathione peroxidase activity compared with the milk from which it has been fermented⁽ Reference Liu, Chen and Lin ^{82 )}. In addition, the free radical-scavenging activity of the plain kefir increased by 10 % with prolonged fermentation from 16 to over 24 h⁽ Reference Liu, Chen and Lin ^{82 )}. However, no difference was observed in the ferrous ion-chelating ability or SOD activity. Liu et al.⁽ Reference Liu, Lin and Chen ^{61 )} explained that ‘The antioxidative activity of kefirs may be attributed to their proton-donating ability, their reducing power and SOD-like activity as evidenced through DPPH and superoxide radical scavenging and lipid peroxidative inhibition results’. Concerning the different types of kefir, Kesenkas et al.⁽ Reference Kesenkaş, Dinkçi and Seçkin ^{83 )} compared full-fat and skimmed milk-based kefir inoculated with grains with a commercial culture using four different antioxidant assays and found that the fat content and the nature of the fermentation did not affect the antioxidant properties of the kefir. These results suggest that kefirs may be relevant candidates for useful and natural antioxidant supplements in the human diet.

Influence of in vitro digestion

Gagnon et al.⁽ Reference Gagnon, Savard and Rivière ^{84 )} evaluated the bioaccessibility of antioxidants during the in vitro digestion of a reconstituted skimmed milk that had been fermented with five selected strains of Bifidobacterium longum ssp. longum. Contrary to previously mentioned studies, the milk fermented with these five strains did not have an initial antioxidant capacity (ORAC assay) higher than unfermented milk. However, higher bioaccessibility of the antioxidants in the fermented milk was observed during digestion. Surprisingly, strains with a low survival rate – compared with the strain showing the best survival rate – in fermented milk were shown to deliver more bioaccessible antioxidants during in vitro dynamic digestion. Therefore, the milk fermented with various strains of Bifidobacterium longum ssp. longum provided bioaccessible antioxidants during digestion, notably at the colonic level, that could prevent the excessive generation of oxidative compounds (i.e. free radicals by microbiota) within the digestive tract⁽ Reference Azcarate-Peril, Sikes and Bruno-Barcena ^{85 )}.

Conclusions

Yoghurt exhibits higher antioxidant potential than milk, notably due to the presence of probiotics generating antioxidant peptides. This is also true for other fermented milks but antioxidant activity greatly depends on the type and number of added probiotics (Table 2).

Influence of cheese processing and composition

The most recent study dealt with the factors affecting the antioxidant capacity (ABTS assay) of 224 different cheese samples (of known composition) prepared using sixteen varied mixtures of milk from cows, ewes and goats during two manufacturing seasons (winter and summer) and over 6 months of ripening⁽ Reference Revilla, Gonzalez-Martin and Vivar-Quintana ^{86 )}. Several correlation analyses were performed. First, the total antioxidant capacity was significantly correlated with the seasonality of manufacturing and the time of ripening but not with the animal species providing the milk. The antioxidant capacity was higher in summer than in winter because of the higher Trolox equivalent antioxidant capacity values observed at month 0 (except for winter goats’ cheese) and mainly at 1 and 2 months in the summer cheeses. This difference resulted from the lower hydrophobic:hydrophilic peptide ratios in summer than in winter cheeses, especially after the first month of ripening, indicating strong proteolysis. Second, significant correlations between the total antioxidant capacity and the retinol content (r 0·399), fat percentage (r 0·308), protein percentage (r 0·366), K content (r 0·385), Mg content (r 0·312), Na content (r 0·432) and P content (r 0·272) were reported, in agreement with previous results⁽ Reference Lucas, Andueza and Rock ^{87 )}. However, the positive correlations between minerals and the total antioxidant capacity are probably due to the significant positive correlation between the protein content (because the total antioxidant capacity is primarily associated with caseins) and these minerals. Based on these results, the authors generated a model to predict the antioxidant capacity in unknown cheeses of different compositions and ripening times⁽ Reference Revilla, Gonzalez-Martin and Vivar-Quintana ^{86 )}.

Therefore, because cheeses exhibit a very high diversity of composition and structure (matrix characteristics), their antioxidant potential could be modulated. Lucas et al.⁽ Reference Lucas, Rock and Chamba ^{88 )} studied the respective effects of the composition of milk and processing on the antioxidant potential of five varieties of French cheese, i.e. Abondance (n 10 cheeses, including both winter and summer cheeses), Tomme de Savoie (n 10, including both winter and summer cheeses), Cantalet (n 12, only winter cheeses) and Salers (n 6, only summer cheeses) from cows’ milk, and Rocamadour (n 15, including both winter and summer cheeses) from goats’ milk. The compositional variability of the cheese in terms of fatty acids, β-carotene, xanthophylls and vitamin E depended mainly on the animal species composition of the milk, i.e. goat v. cow. The total antioxidant capacity of the cheeses ranged from 6·35 to 24·3 mmol Fe²⁺/g DM (FRAP assay). As expected, a higher total antioxidant capacity was measured in goats’ milk. In this study, statistical analyses showed no significant correlation between the milk composition and total antioxidant capacity. Furthermore, none of the antioxidant compounds analysed in this study was found to be positively or negatively correlated with the total antioxidant capacity, which can be explained either by the synergistic action of several compounds or by the action of antioxidant compounds not analysed in this study.

Pattorn & Hongsprabhas⁽ Reference Pattorn and Hongsprabhas ^{89 )} studied the influence of coagulation methods – commonly used to prepare milk protein curds from skimmed milk – on the antioxidant activity of milk protein curds and powders. Precipitation by calcium lactate (curd), coagulation by lactic acid and rennetting of skimmed milk proteins were tested. Curdled milk from precipitation had the densest network (as observed via scanning electron microscopy) and the lowest antioxidant capacity, i.e. 0·27 (ORAC assay) and 0·05 (ABTS assay) μmol TE/mg protein, compared with the other dairy preparations (0·52–0·57 and 0·42–0·44 μmol TE/mg protein, respectively; P<0·05). Furthermore, trypsin hydrolysis for 30 min increased the antioxidant capacity of all products to 0·85–1·11 (ORAC assay) and 1·17–1·19 (ABTS assay) μmol TE/mg protein, regardless of the coagulant used. In terms of practical applications, these results showed that the milk powder precipitated by calcium lactate could be used as a dietary protein source that would provide antioxidant peptides during tryptic digestion. As written by the authors, the ‘study also suggests that the hydrolysates of milk proteins with reasonable antioxidant capacity could be achieved with milk powder prepared from mixed protein coagulated by calcium lactate, lactic acid or rennet, and then hydrolyzed by trypsin’, adding that ‘this is important for developing countries with majority of the population suffering from lactose intolerance, i.e., having milk surplus, yet lacking the capacity of fractionation of milk proteins into individual proteins such as casein β, casein κ, β-lactoglobulin and α-lactalbumin’⁽ Reference Pattorn and Hongsprabhas ^{89 )}.

Gupta et al.⁽ Reference Gupta, Mann and Kumar ^{90 )} aimed to isolate and characterise antioxidant peptides (<3 kDa, isolated by reverse-phase HPLC and analysed by liquid chromatography-tandem MS, ABTS assay) in Cheddar-type cheese inoculated with Lactobacillus casei ssp. casei 300. The authors identified two peptides from milk proteins: VKEAMAPK (m/z 872·5048), corresponding to the 98–105 fraction of β-casein, and HIQKEDVPSER (m/z 1336·7034), corresponding to the 80–90 fraction of α_s1-casein (i.e. protein fragment composed of amino acids initially at positions 98–105 and 80–90 of the linear protein sequence). Interestingly, the antioxidant activity of the VKEAMAPK peptide was comparable with that of commercial antioxidants such as butylated hydroxyanisole, tert-butylhydroquinone and ferulic acid, but the activity of the HIQKEDVPSER peptide was lower. Therefore, this proteolytic strain of lactic acid bacteria can be used to further improve the antioxidant potential of Cheddar cheese via the release of antioxidant peptides.

Influence of ripening time

A recent study investigated the proteolysis and total antioxidant capacity of proteins from white brined cheese prepared from overheated goats’ milk and ripened for 50 d⁽ Reference Barac, Pesic and Zilic ^{91 )}. Proteolysis after cheese ripening induced increases in the antioxidant capacity of both the water-soluble and water-insoluble protein fractions. For example, after 30 d of ripening, the total antioxidant capacity of the water-soluble fraction increased by 12 %. After that period, a more intensive increase was observed, and cheese ripened for 50 d had a total antioxidant capacity that was 56 % higher than that of fresh cheese. This study showed that both the content and nature of N components are essential for the antioxidant capacity. Similarly, Parvin et al. ⁽ Reference Parvin, Rahman and Shimazaki ^{92 )} showed that the antioxidant activity of Bangladesh Gouda and Dhaka cheeses increases with ripening time (up to 2 months).

Gupta et al.⁽ Reference Gupta, Mann and Kumar ^{93 )} evaluated changes in the antioxidant properties (ABTS and DPPH assays, scavenging activity and superoxide anion) of aqueous extracts of Cheddar cheese (inoculated with Lactobacillus casei ssp. casei 300 and Lactobacillus paracasei subsp. paracasei 22) at different stages of ripening but on a longer time scale than in previous studies. As previously reported, the antioxidant activity first increased to a peak at 4 months and then decreased until 9 months of ripening. The changes in the antioxidant activity were linked to the rate of formation of soluble antioxidant peptides upon proteolysis until 4 months. The decrease observed after 4–5 months of ripening indicates that the antioxidant peptides were not resistant to the continuing proteolysis.

The antioxidant capacity (ABTS assay) of water-soluble extracts of Parmigiano Reggiano cheese at different ageing times (up to 41 months) was also studied (ABTS assay)⁽ Reference Bottesini, Paolella and Lambertini ^{94 )}. The antioxidant capacity was determined after simulated in vitro gastrointestinal digestion of these extracts. Contrary to previous studies, the antioxidant capacity in water extracts of Parmigiano Reggiano cheese was unaffected by the ripening time and gastrointestinal digestion. Using liquid chromatography-electrospray ionisation-MS, the authors also showed that the antioxidant capacity of the water-soluble extracts was mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and secondarily to antioxidant peptides, but only minimally.

It is difficult to make generalisations from these studies but it seems that the antioxidant capacity of cheese increases upon ripening to an optimum due to peptide release then decreases due to peptide hydrolysis; this effect and the optimum antioxidant capacity probably depend on cheese type and ripening time.

Influence of in vitro digestion

In the study by Abadia-Garcia et al. ⁽ Reference Abadia-Garcia, Cardador and del Campo ^{95 )}, the viability of probiotic micro-organisms added to cottage cheese in simulated gastrointestinal digestion, the release of peptides with potential antioxidant activity (DPPH and ABTS assays) and their antimicrobial effect on Listeria monocytogenes were evaluated. Cottage cheese was prepared with Lactobacillus casei, Lactobacillus rhamnosus GG, and the commercial mix YO-MIX™ 205 incorporated, in addition to a control without added probiotics. Higher numbers of potentially bioactive peptides were observed in the cheeses with added probiotics. During storage for 28 d (8°C), the antioxidant activity of the soluble cheese extracts determined by the DPPH assay increased, but the one determined by the ABTS assay did not (no difference between the control and the cheeses containing probiotics). Based on the DPPH assay, the increase was strongest with the commercial mix and lower with the control and probiotic Lactobacillus rhamnosus GG. This increase was attributed to the proteolytic activity of the micro-organisms that released antioxidant bioactive peptides. The different behaviour depending on the antioxidant assay used may be due to the different structure of the radical, which can react differently depending on the peptides present in the aqueous extract. According to the authors, these results indicated that the ‘probiotic microorganisms exhibited a significant level of survival in simulated gastric juice when delivered in a cottage cheese’⁽ Reference Abadia-Garcia, Cardador and del Campo ^{95 )}. In contrast, ‘environmental simulated conditions of the duodenum, where bile salts and pancreatic juices are secreted, had more influence on bacterial viability’⁽ Reference Abadia-Garcia, Cardador and del Campo ^{95 )}.

Conclusions

As with yoghurt and fermented milks, cheese also contains antioxidants, especially casein, but with less variety resulting from losses during the cheese-making process. Its total antioxidant activity is mainly determined by the process used, notably that which concentrates the protein fraction leading to higher antioxidant potential than milk. During ripening, proteolysis combined with possible microbial activity may increase this potential (Table 2).