**2. Feeding strategies affecting the bioactive compounds in milk and cheese**

#### **2.1. Fatty acids affected by feeding regimen**

Nutrition is a natural and low-cost way for farmers to rapidly and sharply modulate milk and cheese FA profile towards a healthy profile [24]. The composition of milk fat reflects to some extent the composition of the dietary fat, despite the hydrogenation and isomerisation process to which the FA may be subjected in the rumen. Forages, even though containing a relatively low level of lipids, are often the major source of beneficial unsaturated fatty acids in ruminant diets, and they also provide a low-cost approach to improve milk FA profile in comparison with diet supplementation strategies. In literature, several studies have focused on the impact of different diets on the main milk FA classes, and they also have examined the associations between feeding of various forages and FA composition of milk fat.

Among forages, legumes deserve a special attention due to the raising number of farmers in conversion to organic and low-input production system (i.e. the environmental role of legumes in cropping systems has been even enhanced in Europe by common agricultural policy (CAP) reform) but also to the need of reducing the dependence on the import of protein-rich feed material. Even if, for a given crop, substantial within-species variation occurs, altogether some legume forages such as white clover and birdsfoot trefoil can be considered a rich source of PUFA [25]. Birdsfoot trefoil PUFA content (19.4 g/kg DM) was found higher than in many other legumes, grasses and forbs [26], while white clover with an average ALA content of 16 mg/g DM was a richer source than other common forage legumes (alfalfa, trefoil and red clover) and grasses (orchard grass, fescue and timothy) [27]. Fresh grass is the one main source of ALA. It has been recognised that favourable changes in milk FA profile can be obtained by grazing or feeding fresh forages. Several studies have shown that milk from grazing goats is naturally enriched in fatty acids considered as favourable for human health in comparison to goats fed with high-concentrate diets [24, 28].

Goats unlike sheep are predominantly browsers; in Mediterranean shrublands browse can account for up to 60–80% of goat's diet; animals well adapted to tannin-rich woody forage sources can consume relatively large amounts of tannins without suffering any systemic toxicity [29]. While tannin content in forages is negatively correlated with voluntary intake, digestibility and nitrogen retention, a relatively low amount in ruminant diet can positively affect milk FA composition by protecting dietary PUFA against rumen biohydrogenation [30]. Many forage legumes such as clovers, vetches and Sulla (*Hedysarum coronarium*) are a rich source of polyphenols and especially tannin phenols (TP). Ref. [31] observed that condensed tannins (CT) in Sulla at flowering were contributing to a higher concentration of linoleic and ALA in ewe's milk and a lower ω6/ω3 ratio. Refs. [32, 33] showed that red clover silage, which contained levels of ALA similar to that of grass silage, improved milk PUFA due to a high proportion of red clover ALA passing through the rumen. Polyphenol oxidase (PPO), which is the enzyme involved in the browning reaction of red clover leaves when cut or crushed and exposed to air, has been found to reduce protein and lipid degradations in vitro and potentially in the rumen [34].

Overall, this brief overview on the role of feeding regimen in modulating goat milk fatty acid profile shows that both farm-grown forages including legumes, as well as native pastures, can be considered an effective low-cost way to improve goat milk FA composition without compromising yield and opening new alleys towards a sustainable intensification of the extensive dairy goat system.

#### *2.1.1. Case study: feeding and season on milk fatty acid profile*

This chapter provides an overview of the main bioactive compounds in milk and goat cheese (fatty acids, antioxidant and oligosaccharides) conveying data from significant case studies carried out at the experimental farm of Council for Agricultural Research-Unit for Extensive Husbandry (CREA-ZOE), located in Bella (Muro Lucano, Potenza), Basilicata region (Southern

**2. Feeding strategies affecting the bioactive compounds in milk and** 

between feeding of various forages and FA composition of milk fat.

Nutrition is a natural and low-cost way for farmers to rapidly and sharply modulate milk and cheese FA profile towards a healthy profile [24]. The composition of milk fat reflects to some extent the composition of the dietary fat, despite the hydrogenation and isomerisation process to which the FA may be subjected in the rumen. Forages, even though containing a relatively low level of lipids, are often the major source of beneficial unsaturated fatty acids in ruminant diets, and they also provide a low-cost approach to improve milk FA profile in comparison with diet supplementation strategies. In literature, several studies have focused on the impact of different diets on the main milk FA classes, and they also have examined the associations

Among forages, legumes deserve a special attention due to the raising number of farmers in conversion to organic and low-input production system (i.e. the environmental role of legumes in cropping systems has been even enhanced in Europe by common agricultural policy (CAP) reform) but also to the need of reducing the dependence on the import of protein-rich feed material. Even if, for a given crop, substantial within-species variation occurs, altogether some legume forages such as white clover and birdsfoot trefoil can be considered a rich source of PUFA [25]. Birdsfoot trefoil PUFA content (19.4 g/kg DM) was found higher than in many other legumes, grasses and forbs [26], while white clover with an average ALA content of 16 mg/g DM was a richer source than other common forage legumes (alfalfa, trefoil and red clover) and grasses (orchard grass, fescue and timothy) [27]. Fresh grass is the one main source of ALA. It has been recognised that favourable changes in milk FA profile can be obtained by grazing or feeding fresh forages. Several studies have shown that milk from grazing goats is naturally enriched in fatty acids considered as favourable for human health in comparison to goats fed

Goats unlike sheep are predominantly browsers; in Mediterranean shrublands browse can account for up to 60–80% of goat's diet; animals well adapted to tannin-rich woody forage sources can consume relatively large amounts of tannins without suffering any systemic toxicity [29]. While tannin content in forages is negatively correlated with voluntary intake, digestibility and nitrogen retention, a relatively low amount in ruminant diet can positively affect milk FA composition by protecting dietary PUFA against rumen biohydrogenation [30]. Many forage legumes such as clovers, vetches and Sulla (*Hedysarum coronarium*) are a rich source of polyphenols and especially tannin phenols (TP). Ref. [31] observed that condensed tannins (CT) in Sulla

Italy).

236 Goat Science

**cheese**

**2.1. Fatty acids affected by feeding regimen**

with high-concentrate diets [24, 28].

In this section we report a study carried out at the experimental farm of Council for Agricultural Research and Economics-Research Unit for the Extensive Animal Husbandry (CREA-ZOE) located in Bella (Basilicata region, Southern Italy) during winter, spring and summer seasons. In order to examine changes in milk FA profile under the effect of different feeding regimes, typical of Mediterranean extensive and semi-extensive goat production systems, four groups of Mediterranean Red goats were formed and allocated to different feeding systems: (G) grazing on a native pasture (8 h/day) without supplementation, (GS1) grazing supplemented with 550 g/d of maize and broad beans (CP 14% and NDF 18%, slowly degradable), (GS2) grazing supplemented with 550 g/d of barley and chickpeas (CP 14% and NDF 18%, rapidly degradable) and (H) housing and fed with hay produced with the grass from the same pasture plus 550 g/d of mixed grains (CP 15% and NDF 18%) [35–38]. Regarding lipid extraction method, briefly milk sample (10 ml) was homogenised (2 min) with CHCl<sup>3</sup> and MeOH mixture (2/1, v/v) and centrifuged (500 × g, 10 min). After removing the upper layer, the lower layer was filtered through a Buchner funnel, rinsed with CHCl<sup>3</sup> (30 ml) and then again filtered. The chloroformlipid extract was dried over anhydrous Na<sup>2</sup> SO<sup>4</sup> , rinsed with CHCl<sup>3</sup> (30 ml) and concentrated using a rotary evaporator at 30°C. The residue was stored at −80°C for lipid determination. Lipid extract was methylated adding hexane (1 ml) and 2 N methanolic KOH (0.05 ml). Gas chromatograph analysis was performed on a Varian model 3800 GC instrument fitted with an automatic sampler (CP 8410) for a multiple injection. Fatty acid methyl esters (FAME) were separated through a cyanopropyl polysiloxane (DB 23, J & W) fused silica capillary column (60 m × 0.25 mm i.d.). Operating conditions were a helium flow rate of 1.2 ml/min, a FID detector at 250°C and a split-splitless injector at 230°C with a split ratio 1:100. The column temperature was held at 60°C for 5 min after sample injection (1 μl), increased at 14°C/min to 165°C and at 2°C/min to 225°C and held at 225°C for 20 min. The individual fatty acid peaks were identified with reference to the retention times of standard of CLA isomers (cis-9, trans-11 97% and trans-10, cis-12 3%; Larodan, Malmö, Sweden) and a known mixture of standards (FAME, Sigma). Fatty acids were expressed as percentage of total FAME.

Milk produced by goat groups showed a wide variability in its FA composition linked to the characteristics of the ingested feed in each type of feeding system (**Figure 1**). In particular,

**Figure 1.** Effect of feeding system × season interaction on vaccenic acid (VA), linoleic acid (LA), conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) content and on Δ9-desaturase activity (C14:1/C14:0) of milk of goats fed with (G) grazing on native pasture without supplementation, (GS1) grazing plus maize and broad beans (GS2) and grazing plus barley and chickpeas and (H) housed and fed with hay plus mixed grains (modified from Refs. [35–37]). a, b and c = *P* < 0.05.

G and GS1 groups produced milk with a higher content of CLA and VA compared to other groups. Indeed, it is noted that the consumption of high-concentrate diets, compared with high-forage ones, affects the extent of ruminal biohydrogenation with a consequent reduction of CLA and VA production. The high variability of CLA and VA levels in milk of G group, with the highest level reached in winter, could be ascribed to the seasonal changes in grass availability and in phenological stage of the plants. The similar pattern of VA and CLA observed in milk of goat rearing in different feeding systems confirms the positive relationship between these intermediate products of ruminal biohydrogenation. Feeding regimen and season also affected Δ9 -desaturase activity (C14:1/C14:0), responsible of endogenous synthesis of CLA, with fresh grass being able to enhance this enzyme activity (**Figure 1**).

The effect of different feeding systems on beneficial FA in milk is more evident using the health-promoting index (HPI, **Figure 2**), an index that expresses the health value of dietary fat, and it is calculated as follows: total unsaturated FA/[C12:0 + (4 × C14:0) + C16:0] [41]. Dairy product with high HPI value is assumed to be more beneficial to human health. According to this index, pasture feeding allows the optimisation of the balance between detrimental and valuable fatty acids in goat milk, thus obtaining beneficial effects

**Figure 2.** Effect of feeding system × season interaction on n-6/n-3 and health-promoting index (HPI) value of milk of goats fed with (G) grazing on native pasture without supplementation, (GS1) grazing plus maize and broad beans and (GS2) grazing plus barley and chickpeas and (H) housed and fed with hay plus mixed grains [modified from Ref. [38]).

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The results of this study show that milk from goats fed with pasture had higher amounts of nutritionally peculiar FA than milk from other feeding treatments. On the other hand, grazing supplementation with concentrates that better interact with the nutritive characteristics of pasture could represent a strategy to meet nutritional requirements of animals and sustain

In this section we report a study carried out at the CREA-ZOE experimental farm in spring. Eight homogeneous groups of Red Syrian goats have been allocated to eight different feeding treatments. The housed goat groups received during 11 days a single forage species *ad libitum*, and they had access to water and salt blocks. Daily, the forages were cut and provided to the goat groups. Seven forages (*Avena sativa*, *Lolium perenne*, *Hordeum vulgare*, *Lotus corniculatus*, *Medicago sativa*, *Trifolium incarnatum*, *Vicia sativa*) were used at the phenological stage commonly used in Southern Italy for grazing and one (*Triticosecale*) as silage. After an adaptation period to the forage

for consumer's health.

milk production without worsening its quality.

A, B and C = *P* < 0.01 for n6/n3. a, b and c = *P* < 0.05 for HPI.

*2.1.2. Case study: relationship between forage species and fatty acids of cheese*

Grazing significantly increased the proportion of long-chain n-3 PUFA, such as DHA, and decreased the n-6/n-3 PUFA ratio in milk (**Figure 2**). The level of DHA reached interesting value in milk fat of grazing goats in winter probably because of the high content of its precursor (ALA) in the pasture. The ratio between n-3 PUFA and n-6 PUFA is an index commonly used to assess the nutritional value of fats [39]. Housing goats exhibited a higher n-6/n-3 PUFA ratio than other treatments, probably attributable to the high level of LA in milk (**Figure 1**), the main component of n-6 PUFA. The composition of concentrate mixture offered to H group appears to explain the highest content of LA found in milk fat.

The distribution of concentrates to grazing goats significantly affected milk FA profile. Under grazing condition, GS1 dietary treatment characterised by slowly degradable concentrate improved milk FA profile compared to GS2 group fed with rapidly degradable concentrate. Probably the supplementation received by GS1 group could have determined a rumen environment favourable to a less efficient biohydrogenation of substrate with consequent accumulation of intermediate products. Besides, the differences observed in milk FA composition between supplemented and non-supplemented grazing groups could be linked to the different herbage selections of supplemented grazing goats, as suggested in Ref. [40].

Bioactive Compounds in Goat Milk and Cheese: The Role of Feeding System and Breed http://dx.doi.org/10.5772/intechopen.70083 239

**Figure 2.** Effect of feeding system × season interaction on n-6/n-3 and health-promoting index (HPI) value of milk of goats fed with (G) grazing on native pasture without supplementation, (GS1) grazing plus maize and broad beans and (GS2) grazing plus barley and chickpeas and (H) housed and fed with hay plus mixed grains [modified from Ref. [38]). A, B and C = *P* < 0.01 for n6/n3. a, b and c = *P* < 0.05 for HPI.

The effect of different feeding systems on beneficial FA in milk is more evident using the health-promoting index (HPI, **Figure 2**), an index that expresses the health value of dietary fat, and it is calculated as follows: total unsaturated FA/[C12:0 + (4 × C14:0) + C16:0] [41]. Dairy product with high HPI value is assumed to be more beneficial to human health. According to this index, pasture feeding allows the optimisation of the balance between detrimental and valuable fatty acids in goat milk, thus obtaining beneficial effects for consumer's health.

The results of this study show that milk from goats fed with pasture had higher amounts of nutritionally peculiar FA than milk from other feeding treatments. On the other hand, grazing supplementation with concentrates that better interact with the nutritive characteristics of pasture could represent a strategy to meet nutritional requirements of animals and sustain milk production without worsening its quality.

#### *2.1.2. Case study: relationship between forage species and fatty acids of cheese*

G and GS1 groups produced milk with a higher content of CLA and VA compared to other groups. Indeed, it is noted that the consumption of high-concentrate diets, compared with high-forage ones, affects the extent of ruminal biohydrogenation with a consequent reduction of CLA and VA production. The high variability of CLA and VA levels in milk of G group, with the highest level reached in winter, could be ascribed to the seasonal changes in grass availability and in phenological stage of the plants. The similar pattern of VA and CLA observed in milk of goat rearing in different feeding systems confirms the positive relationship between these intermediate products of ruminal biohydrogenation. Feeding regimen and season also

**Figure 1.** Effect of feeding system × season interaction on vaccenic acid (VA), linoleic acid (LA), conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) content and on Δ9-desaturase activity (C14:1/C14:0) of milk of goats fed with (G) grazing on native pasture without supplementation, (GS1) grazing plus maize and broad beans (GS2) and grazing plus barley and chickpeas and (H) housed and fed with hay plus mixed grains (modified from Refs. [35–37]). a, b and c = *P* < 0.05.

Grazing significantly increased the proportion of long-chain n-3 PUFA, such as DHA, and decreased the n-6/n-3 PUFA ratio in milk (**Figure 2**). The level of DHA reached interesting value in milk fat of grazing goats in winter probably because of the high content of its precursor (ALA) in the pasture. The ratio between n-3 PUFA and n-6 PUFA is an index commonly used to assess the nutritional value of fats [39]. Housing goats exhibited a higher n-6/n-3 PUFA ratio than other treatments, probably attributable to the high level of LA in milk (**Figure 1**), the main component of n-6 PUFA. The composition of concentrate mixture offered to H group

The distribution of concentrates to grazing goats significantly affected milk FA profile. Under grazing condition, GS1 dietary treatment characterised by slowly degradable concentrate improved milk FA profile compared to GS2 group fed with rapidly degradable concentrate. Probably the supplementation received by GS1 group could have determined a rumen environment favourable to a less efficient biohydrogenation of substrate with consequent accumulation of intermediate products. Besides, the differences observed in milk FA composition between supplemented and non-supplemented grazing groups could be linked to the differ-

ent herbage selections of supplemented grazing goats, as suggested in Ref. [40].

with fresh grass being able to enhance this enzyme activity (**Figure 1**).

appears to explain the highest content of LA found in milk fat.


affected Δ9

238 Goat Science

In this section we report a study carried out at the CREA-ZOE experimental farm in spring. Eight homogeneous groups of Red Syrian goats have been allocated to eight different feeding treatments. The housed goat groups received during 11 days a single forage species *ad libitum*, and they had access to water and salt blocks. Daily, the forages were cut and provided to the goat groups. Seven forages (*Avena sativa*, *Lolium perenne*, *Hordeum vulgare*, *Lotus corniculatus*, *Medicago sativa*, *Trifolium incarnatum*, *Vicia sativa*) were used at the phenological stage commonly used in Southern Italy for grazing and one (*Triticosecale*) as silage. After an adaptation period to the forage supplied, the milk of each group was collected and processed into *Caciotta* cheese, a traditional goat cheese manufactured in Southern Italy, ripened for 20 days. Cheese samples (3 g) were finely grated, and lipid extraction and composition were performed as described in Section 2.1.1.

In this study, forage species had an effect on FA profile of *Caciotta* cheese [42]. As regards CLA (**Figure 3**), cheeses from goat groups fed with *T. incarnatum*, *Triticosecale* and *H. vulgare* showed higher values than those obtained from *A. sativa* and *L. corniculatus*. The lowest content of CLA and the highest content of ALA were detected in the cheese made from milk of goats fed with *V. sativa*. Cheeses from *L. corniculatus* and *M. sativa* displayed the same ALA content. The ALA showed a major variation among cheeses (range 1.04), while the CLA exhibited a smaller variation (range 0.349) [42–44].

Cheeses from legume groups showed significantly higher values of ALA compared with grass groups, whereas the content of ALA in cheese from *Triticosecale* silage was in trend with other fresh grasses. Green fodders are excellent source of ALA and are the most effective feeds in shifting the milk FA profile towards a healthy profile. Fortunately, the milk processing does not change substantially its FA profile [45]; it follows that the bioactive compounds, CLA and ALA, of dairy products are dependent of their content in the unprocessed raw milk [43, 46]. The effects of different forage species on ALA, VA and CLA content in cheeses could be connected to the high content of PUFA in green forage, with ALA being the most representative of this FA class (**Figure 4**), and to the role of secondary metabolites (polyphenols) and vegetable enzymes (polyphenol oxidase) present in forage species. These compounds have potential to interact with lipolysis and biohydrogenation of PUFA in vitro, in fact a negative relationship was found between tannic polyphenols/ALA content ratio and ALA biohydrogenation [47]. In our study, the highest content of ALA in cheese from goats fed with *V. sativa* could be linked to the higher level of tannic polyphenols in the forage as reported by Ref. [48].

The health-promoting index was calculated in order to have an immediate view of the bioactive compounds present in the cheese [41]. The forage species affected the HPI (**Figure 5**). The higher HPI values observed in *Caciotta* cheese from goat groups fed *H. vulgare* and *A. sativa* [43, 44] could be linked to the high level of PUFA found in these forages (**Figure 4**). The HPI observed in other cheeses is still higher compared to those found in milk from animals fed

**Figure 4.** Comparison of percentage content of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and

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241

polyunsaturated fatty acids (PUFA) in fresh forage species (authors' own unpublished data).

This case study shows that the single forage characterises the bioactive compounds' content in cheese; this result can be a strategy to guide, depending on farm fodder availability, the

Measuring the total antioxidant capacity of milk and cheese helps to understand the relationships between the bioactive compounds present in milk and their ability to protect the substrate. Antioxidant activity can be enhanced by providing food as a source of antioxidant components [16]. The results of a few recent studies show the accumulation of various bioactive compounds biotransformed and/or as such in the milk and cheese of grazing goats or fed with a mixture of forage legume [18, 49]. The high value of total phenolic concentration with added nutritional and sensory values, without changing properties of the cheeses itself, was observed in cheeses made from goats fed with non-distilled thyme leaves, one of the aromatic

with dry fodder (see Section 2.1.1).

**2.2. Antioxidant compounds in goat milk**

production of dairy products beneficial to human health.

**Figure 3.** Comparison of conjugated linoleic acid (CLA) and α-linolenic acid (ALA) of Caciotta cheese made from milk of goats fed *ad libitum* with a single forage species (modified from Refs. [42–44]). a–d = *P* < 0.05.

**Figure 4.** Comparison of percentage content of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in fresh forage species (authors' own unpublished data).

The health-promoting index was calculated in order to have an immediate view of the bioactive compounds present in the cheese [41]. The forage species affected the HPI (**Figure 5**). The higher HPI values observed in *Caciotta* cheese from goat groups fed *H. vulgare* and *A. sativa* [43, 44] could be linked to the high level of PUFA found in these forages (**Figure 4**). The HPI observed in other cheeses is still higher compared to those found in milk from animals fed with dry fodder (see Section 2.1.1).

This case study shows that the single forage characterises the bioactive compounds' content in cheese; this result can be a strategy to guide, depending on farm fodder availability, the production of dairy products beneficial to human health.

#### **2.2. Antioxidant compounds in goat milk**

supplied, the milk of each group was collected and processed into *Caciotta* cheese, a traditional goat cheese manufactured in Southern Italy, ripened for 20 days. Cheese samples (3 g) were finely grated, and lipid extraction and composition were performed as described in Section 2.1.1. In this study, forage species had an effect on FA profile of *Caciotta* cheese [42]. As regards CLA (**Figure 3**), cheeses from goat groups fed with *T. incarnatum*, *Triticosecale* and *H. vulgare* showed higher values than those obtained from *A. sativa* and *L. corniculatus*. The lowest content of CLA and the highest content of ALA were detected in the cheese made from milk of goats fed with *V. sativa*. Cheeses from *L. corniculatus* and *M. sativa* displayed the same ALA content. The ALA showed a major variation among cheeses (range 1.04), while the CLA exhib-

Cheeses from legume groups showed significantly higher values of ALA compared with grass groups, whereas the content of ALA in cheese from *Triticosecale* silage was in trend with other fresh grasses. Green fodders are excellent source of ALA and are the most effective feeds in shifting the milk FA profile towards a healthy profile. Fortunately, the milk processing does not change substantially its FA profile [45]; it follows that the bioactive compounds, CLA and ALA, of dairy products are dependent of their content in the unprocessed raw milk [43, 46]. The effects of different forage species on ALA, VA and CLA content in cheeses could be connected to the high content of PUFA in green forage, with ALA being the most representative of this FA class (**Figure 4**), and to the role of secondary metabolites (polyphenols) and vegetable enzymes (polyphenol oxidase) present in forage species. These compounds have potential to interact with lipolysis and biohydrogenation of PUFA in vitro, in fact a negative relationship was found between tannic polyphenols/ALA content ratio and ALA biohydrogenation [47]. In our study, the highest content of ALA in cheese from goats fed with *V. sativa* could be linked to

**Figure 3.** Comparison of conjugated linoleic acid (CLA) and α-linolenic acid (ALA) of Caciotta cheese made from milk of

goats fed *ad libitum* with a single forage species (modified from Refs. [42–44]). a–d = *P* < 0.05.

the higher level of tannic polyphenols in the forage as reported by Ref. [48].

ited a smaller variation (range 0.349) [42–44].

240 Goat Science

Measuring the total antioxidant capacity of milk and cheese helps to understand the relationships between the bioactive compounds present in milk and their ability to protect the substrate. Antioxidant activity can be enhanced by providing food as a source of antioxidant components [16]. The results of a few recent studies show the accumulation of various bioactive compounds biotransformed and/or as such in the milk and cheese of grazing goats or fed with a mixture of forage legume [18, 49]. The high value of total phenolic concentration with added nutritional and sensory values, without changing properties of the cheeses itself, was observed in cheeses made from goats fed with non-distilled thyme leaves, one of the aromatic

21 mg/kg dry matter, respectively) [26]. In forages, however, the complex mechanisms of interaction between pro- and antioxidant compounds must be taken into account [55]. While legumes might not be the richest/most effective source of α-tocopherol, it is the synergy between the phenolic acids, CT and anthocyanins that contribute to build up the so-called antioxidant network [56]. Altogether, the choice of natural feeding strategies for goats, without the use of expensive supplements, synthetic and/or encapsulated, could provide a way to encourage the consumer to the choice of dairy products obtained with natural resources and

Bioactive Compounds in Goat Milk and Cheese: The Role of Feeding System and Breed

Mediterranean pastures are highly variable in relation to the season, the proportion of grass plants decreases from 85 to 55% from winter to spring, while forbs increase from 25 to 65% from late spring to early summer. In early summer, goats graze mainly on forbs, some of which are used as medical plants by human. In order to highlight a relationship between non-volatile phenolic compounds in plant species and the same class of metabolites in milk or cheese, Ref. [14] examined the nuclear magnetic resonance (NMR) spectra of two green plants, borage (*Borago officinalis* L.) and hawthorn (*Crataegus oxyacantha* L.) (**Figure 6**) and milk (**Figure 7**) obtained from two groups of goats fed *ad libitum* with these plants during 18 days. A control group was fed *ad libitum* with natural hay and concentrate. Briefly, air-dried plants (500 g) were extracted with

and MeOH (10%, w/v) at room temperature. Chloroform extracts were fractionated on

residue, which was chromatographed on Sephadex LH-20 eluting with MeOH. Fractions were purified by RP-HPLC as reported above. Milk sample (1 L) was lyophilised and then extracted, fractionated and purified as described for plant sample. The structure of the pure compounds

DRX 600 NMR Spectrometer; Bruker, Karlsruhe, Germany) and by comparison with literature data. Compound identification was also confirmed, when possible, by HPLC analyses with ref-

The authors found a relationship between the antioxidant intake from borage and hawthorn and the levels of antioxidant metabolites in milk, flavonoids and terpenoids contained in these herbs that were found in milk. Quercetin and rutin were excreted in part without modification, while other compounds were structurally modified. No metabolite has been found in the control group milk. The different solvents, methanol or chloroform, used in the complex method of extraction for the plant material and milk have generated great differences in the recovered metabolites. For the purpose of a useful comparison of results from different

Thus, the hypothesis of the authors was that gastrointestinal microflora of goats can structurally modify plant metabolites through hydrolyses and/or other interactions that result in structurally less complex molecules in milk. This study demonstrates that the presence of

ity. Fractions were purified by RP-HPLC on a μ-Bondapak column eluting with H<sup>2</sup>

(1:1). Methanol extracts of plants were fractionated between BuOH and H<sup>2</sup>

erence to the retention times of standards (Sigma-Aldrich Co., Milan).

experiments, the standardization of extraction methods appears to be desirable.

and CHCI<sup>3</sup>


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243

H, 13C and 13C DEPT NMR data (Bruker

O-MeOH

O to give a butanol

associated with beneficial health effects beyond its pure nutritional value.

*2.2.1. Case study 1: borage and hawthorn and phenolic compounds in milk*

silica-gel column (80 × 4 cm) eluting with CHCI<sup>3</sup>

isolated from samples was determined by analysis of 1

phenolic compounds in milk depends on the animal feed.

CHCl<sup>3</sup>

**Figure 5.** Comparison of health-promoting index (HPI) of *Caciotta* cheese made from milk of goats fed *ad libitum* with a single forage species (modified from Refs. [43, 44]).

plants widespread in the Mediterranean area [50]. The influence of high-polyphenol diet on cheese total polyphenol content and antioxidant capacity has been reported by Ref. [51]. They found higher level of polyphenolic compounds and antioxidant activity in cheese produced with milk from grazing goats, with a rich content of secondary metabolites, in comparison with cheese from goats kept in full indoor confinement and fed with Lucerne hay and concentrate. Grazing management represents a better option than indoor feeding to produce a healthy profile of bioactive compounds, providing an increase of total polyphenol, hydroxycinnamic acids and flavonoid concentrations. The feeding strategy involves not only polyphenols but also fat-soluble vitamins, especially those that play an important role as antioxidant (α-tocopherol and β-carotene). A positive relationship was observed between pasture-based rations rather than the hay-based rations for goats and levels of α-tocopherol and retinol in Rocamadour cheese, while β-carotene was not detected [52]. Ref. [49] found that grazing level high and medium, as percentage of net energy of requirement recovered from pasture, on Mediterranean shrublands and month of grazing also affect α-tocopherol content in goat milk without change of milk total antioxidant capacity.

Among forages, legumes are a rich source of polyphenols; large variability occurs among species; some ancient crops like common vetch (*V. sativa*) were found to contain threefold more polyphenol and five times more flavonoids than soybean [53]. Another important class of antioxidant compounds in legumes is represented by carotenoids and tocopherols; as many other compounds, their concentration is influenced by leaf proportion. Ref. [54] recommend a strategic approach to the choice of harvest date and wilting duration, since these can be a key tool for manipulating vitamins and FA composition in forages. Among forage legumes birdsfoot trefoil shows a relatively high content of lutein and contains almost threefold higher concentration of α-tocopherol content than yellow sweet clover and Lucerne (65 vs. 23 and 21 mg/kg dry matter, respectively) [26]. In forages, however, the complex mechanisms of interaction between pro- and antioxidant compounds must be taken into account [55]. While legumes might not be the richest/most effective source of α-tocopherol, it is the synergy between the phenolic acids, CT and anthocyanins that contribute to build up the so-called antioxidant network [56]. Altogether, the choice of natural feeding strategies for goats, without the use of expensive supplements, synthetic and/or encapsulated, could provide a way to encourage the consumer to the choice of dairy products obtained with natural resources and associated with beneficial health effects beyond its pure nutritional value.

#### *2.2.1. Case study 1: borage and hawthorn and phenolic compounds in milk*

plants widespread in the Mediterranean area [50]. The influence of high-polyphenol diet on cheese total polyphenol content and antioxidant capacity has been reported by Ref. [51]. They found higher level of polyphenolic compounds and antioxidant activity in cheese produced with milk from grazing goats, with a rich content of secondary metabolites, in comparison with cheese from goats kept in full indoor confinement and fed with Lucerne hay and concentrate. Grazing management represents a better option than indoor feeding to produce a healthy profile of bioactive compounds, providing an increase of total polyphenol, hydroxycinnamic acids and flavonoid concentrations. The feeding strategy involves not only polyphenols but also fat-soluble vitamins, especially those that play an important role as antioxidant (α-tocopherol and β-carotene). A positive relationship was observed between pasture-based rations rather than the hay-based rations for goats and levels of α-tocopherol and retinol in Rocamadour cheese, while β-carotene was not detected [52]. Ref. [49] found that grazing level high and medium, as percentage of net energy of requirement recovered from pasture, on Mediterranean shrublands and month of grazing also affect α-tocopherol content in goat milk

**Figure 5.** Comparison of health-promoting index (HPI) of *Caciotta* cheese made from milk of goats fed *ad libitum* with a

Among forages, legumes are a rich source of polyphenols; large variability occurs among species; some ancient crops like common vetch (*V. sativa*) were found to contain threefold more polyphenol and five times more flavonoids than soybean [53]. Another important class of antioxidant compounds in legumes is represented by carotenoids and tocopherols; as many other compounds, their concentration is influenced by leaf proportion. Ref. [54] recommend a strategic approach to the choice of harvest date and wilting duration, since these can be a key tool for manipulating vitamins and FA composition in forages. Among forage legumes birdsfoot trefoil shows a relatively high content of lutein and contains almost threefold higher concentration of α-tocopherol content than yellow sweet clover and Lucerne (65 vs. 23 and

without change of milk total antioxidant capacity.

single forage species (modified from Refs. [43, 44]).

242 Goat Science

Mediterranean pastures are highly variable in relation to the season, the proportion of grass plants decreases from 85 to 55% from winter to spring, while forbs increase from 25 to 65% from late spring to early summer. In early summer, goats graze mainly on forbs, some of which are used as medical plants by human. In order to highlight a relationship between non-volatile phenolic compounds in plant species and the same class of metabolites in milk or cheese, Ref. [14] examined the nuclear magnetic resonance (NMR) spectra of two green plants, borage (*Borago officinalis* L.) and hawthorn (*Crataegus oxyacantha* L.) (**Figure 6**) and milk (**Figure 7**) obtained from two groups of goats fed *ad libitum* with these plants during 18 days. A control group was fed *ad libitum* with natural hay and concentrate. Briefly, air-dried plants (500 g) were extracted with CHCl<sup>3</sup> and MeOH (10%, w/v) at room temperature. Chloroform extracts were fractionated on silica-gel column (80 × 4 cm) eluting with CHCI<sup>3</sup> and CHCI<sup>3</sup> -MeOH mixture of increasing polarity. Fractions were purified by RP-HPLC on a μ-Bondapak column eluting with H<sup>2</sup> O-MeOH (1:1). Methanol extracts of plants were fractionated between BuOH and H<sup>2</sup> O to give a butanol residue, which was chromatographed on Sephadex LH-20 eluting with MeOH. Fractions were purified by RP-HPLC as reported above. Milk sample (1 L) was lyophilised and then extracted, fractionated and purified as described for plant sample. The structure of the pure compounds isolated from samples was determined by analysis of 1 H, 13C and 13C DEPT NMR data (Bruker DRX 600 NMR Spectrometer; Bruker, Karlsruhe, Germany) and by comparison with literature data. Compound identification was also confirmed, when possible, by HPLC analyses with reference to the retention times of standards (Sigma-Aldrich Co., Milan).

The authors found a relationship between the antioxidant intake from borage and hawthorn and the levels of antioxidant metabolites in milk, flavonoids and terpenoids contained in these herbs that were found in milk. Quercetin and rutin were excreted in part without modification, while other compounds were structurally modified. No metabolite has been found in the control group milk. The different solvents, methanol or chloroform, used in the complex method of extraction for the plant material and milk have generated great differences in the recovered metabolites. For the purpose of a useful comparison of results from different experiments, the standardization of extraction methods appears to be desirable.

Thus, the hypothesis of the authors was that gastrointestinal microflora of goats can structurally modify plant metabolites through hydrolyses and/or other interactions that result in structurally less complex molecules in milk. This study demonstrates that the presence of phenolic compounds in milk depends on the animal feed.

**Figure 6.** Metabolites found in plant of *Borago officinalis* and *Crataegus oxyacantha*. Plant extract with methanol (a) and chloroform (b) (modified from Ref. [14]).

and 3 days of experiment and sample collection, phenolic compounds were extracted from herbage, milk, whey and cheeses by methods appropriate to the substrate and analysed by

**Figure 7.** Plant metabolites found in milk from goats fed with *Borago officinalis* and *Crataegus oxyacantha*. Milk extract

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Ten Siriana goats were fed indoor with *A. sativa* in pureness, given fresh for 10 days of adaptation and 3 days of trial. In these 3 days, herbage (the part of plant effectively ingested) was daily collected, freeze-dried and ball milled. Milk and whey samples were collected on days 2 and 3, contextually with cheesemaking (*Caciotta* cheese) and stored at −20°C.

O (80:20)

CN (22 ml)

Briefly, phenolic compounds were extracted from herbage (200 mg) with EtOH/H<sup>2</sup>

at 90°C while from milk (10 ml) and whey (10 ml) by a precipitation in CH<sup>3</sup>

high-performance liquid chromatography (HPLC-DAD).

with methanol (a) and chloroform (b) (modified from Ref. [14]).

#### *2.2.2. Case study 2: oat and phenolic compounds*

The wild species or aromatic plants in the pasture are less present in quantity than forage species. As forages represent a high proportion of ruminant diet, in order to observe the link between phenolic content of forage species and phenolic content of milk, whey and cheese, Ref. [15] planned an experiment with ten Mediterranean Red goats fed indoor with fresh *A. sativa* forage, in pureness, without any other supplementation. After 10 days of adaptation

**Figure 7.** Plant metabolites found in milk from goats fed with *Borago officinalis* and *Crataegus oxyacantha*. Milk extract with methanol (a) and chloroform (b) (modified from Ref. [14]).

and 3 days of experiment and sample collection, phenolic compounds were extracted from herbage, milk, whey and cheeses by methods appropriate to the substrate and analysed by high-performance liquid chromatography (HPLC-DAD).

*2.2.2. Case study 2: oat and phenolic compounds*

chloroform (b) (modified from Ref. [14]).

244 Goat Science

The wild species or aromatic plants in the pasture are less present in quantity than forage species. As forages represent a high proportion of ruminant diet, in order to observe the link between phenolic content of forage species and phenolic content of milk, whey and cheese, Ref. [15] planned an experiment with ten Mediterranean Red goats fed indoor with fresh *A. sativa* forage, in pureness, without any other supplementation. After 10 days of adaptation

**Figure 6.** Metabolites found in plant of *Borago officinalis* and *Crataegus oxyacantha*. Plant extract with methanol (a) and

Ten Siriana goats were fed indoor with *A. sativa* in pureness, given fresh for 10 days of adaptation and 3 days of trial. In these 3 days, herbage (the part of plant effectively ingested) was daily collected, freeze-dried and ball milled. Milk and whey samples were collected on days 2 and 3, contextually with cheesemaking (*Caciotta* cheese) and stored at −20°C. Briefly, phenolic compounds were extracted from herbage (200 mg) with EtOH/H<sup>2</sup> O (80:20) at 90°C while from milk (10 ml) and whey (10 ml) by a precipitation in CH<sup>3</sup> CN (22 ml) and an overnight deconjugation using a glucuronidase-sulfatase enzyme mixture. Cheese was homogenised and centrifuged, and the supernatant was used for phenolic extraction as described for milk. Phenolic compounds of the extracts were analysed as UV-absorbing compounds using HPLC-DAD on a reverse phase column (LiChroCART® 125-4, Merck) eluted by 0.3 ml/min of a 0–100% gradient of acetonitrile in water, both containing 0.1% formic acid. The UV spectra were compared to those of standard compounds and classified into simple phenol, benzoic acid derivatives, cinnamic acid derivative and flavones groups. The *A. sativa* forage revealed an interesting metabolite profile, where cinnamic acid and flavones largely occur (mostly derived from apigenin and luteolin or chrysoeriol). These phenolic compounds affected milk and whey, even though in different measures: flavones disappeared, while simple phenols, benzoic acid derivatives and some unclassified phenols were identified (**Figure 8**).

In cheese, although the largest amount of phenolic compounds was still by benzoic acid derivatives, there was a greater number of simple phenols and one of the indole derivatives found in milk. Nevertheless, phenolic compounds' profile of milk was much closer to whey profile than

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These preliminary results have allowed us to get an overview of the transfer of the plant metabolites directly or processed or degraded in the digestive tract, to the product. However, quantitative studies would be desirable to measure the partition of phenolic compounds in

In goat feeding, forage plants such as grasses and legumes have an essential role, since they represent a high proportion of diet. Forages commonly used in Mediterranean area can be a natural source of bioactive compounds that can be transferred to animal products. In order to evaluate and compare the potential contribution of some grass and legume species, to increase the level of bioactive compounds and antioxidant capacity in milk, Refs. [48, 57] compared the total polyphenol intake of three grasses, *Festuca arundinacea*, *H. vulgare* and *Triticosecale*, and four legumes, *Pisum sativum*, *Trifolium alexandrinum*, *V. sativa* and *Vicia faba minor*, given to seven groups of Mediterranean Red goats without supplement for eleven days. The single forage was cut daily and given *ad libitum* indoor. After an adaptation period, forage samples and milk samples of each group were collected and analysed for polyphenolic compounds and total antioxidant capacity. Folin-Ciocalteu method as described by Ref. [60] was used to determine tannic and non-tannic polyphenol contents in forage samples, after the addition of insoluble matrix polyvinylpolypyrrolidone (PVPP) and total and free polyphenol contents in milk samples. Contents were expressed as gallic acid equivalents (GAE). Milk conjugate polyphenol content was obtained by difference between milk total and free polyphenol contents. Total antioxidant capacity (TAC) was measured using the ferric-reducing antioxidant power

*2.2.3. Case study 3: relationship between forage species and antioxidant compounds in milk*

as indicated by Benzie and Strain method and was expressed as μM FeSO<sup>4</sup>

as such, to milk according to De Feo et al.'s [14] study.

phenol content (**Figure 9**) and the antioxidant capacity (**Figure 10**), respectively.

*2.2.4. Case study 4: Sulla forage and phenolic compounds and antioxidant capacity*

Among forages, *T. alexandrinum* and *F. arundinacea* were shown to enhance the milk-free poly-

The phenological stage to which the fodder was used by goats may have contributed, as reported in the literature [47], to total polyphenol intake (**Figure 9**) and milk polyphenol content. As polyphenolic beneficial compounds occur largely in forages, it could be assumed their possible relationships and their transfer from diet, through some biotransformations or,

Among plant species that are used in ruminant feed in the Mediterranean area, Sulla (*Sulla coronarium* L.), which is a short-lived perennial legume, plays a key role in the cereal-based systems that are used in semiarid regions. This legume forage has widespread availability in Mediterranean areas, where it is greatly appreciated for the positive effects of its nutrient and CT contents on milk yield and composition, as demonstrated in both sheep and goats [58, 59].

.

to cheese ones.

serum and cheese.

**Figure 8.** Phenolic compounds in *Avena sativa* forage and milk, whey and cheese from goats fed with fresh *Avena sativa* in pureness. (a) Data are expressed as abundance of compounds and (b) percentage of total picks by HPLC-DAD analyses (modified from Ref. [48]).

In cheese, although the largest amount of phenolic compounds was still by benzoic acid derivatives, there was a greater number of simple phenols and one of the indole derivatives found in milk. Nevertheless, phenolic compounds' profile of milk was much closer to whey profile than to cheese ones.

and an overnight deconjugation using a glucuronidase-sulfatase enzyme mixture. Cheese was homogenised and centrifuged, and the supernatant was used for phenolic extraction as described for milk. Phenolic compounds of the extracts were analysed as UV-absorbing compounds using HPLC-DAD on a reverse phase column (LiChroCART® 125-4, Merck) eluted by 0.3 ml/min of a 0–100% gradient of acetonitrile in water, both containing 0.1% formic acid. The UV spectra were compared to those of standard compounds and classified into simple phenol, benzoic acid derivatives, cinnamic acid derivative and flavones groups. The *A. sativa* forage revealed an interesting metabolite profile, where cinnamic acid and flavones largely occur (mostly derived from apigenin and luteolin or chrysoeriol). These phenolic compounds affected milk and whey, even though in different measures: flavones disappeared, while simple phenols, benzoic acid derivatives and some unclassified phenols

**Figure 8.** Phenolic compounds in *Avena sativa* forage and milk, whey and cheese from goats fed with fresh *Avena sativa* in pureness. (a) Data are expressed as abundance of compounds and (b) percentage of total picks by HPLC-DAD analyses

were identified (**Figure 8**).

246 Goat Science

(modified from Ref. [48]).

These preliminary results have allowed us to get an overview of the transfer of the plant metabolites directly or processed or degraded in the digestive tract, to the product. However, quantitative studies would be desirable to measure the partition of phenolic compounds in serum and cheese.

#### *2.2.3. Case study 3: relationship between forage species and antioxidant compounds in milk*

In goat feeding, forage plants such as grasses and legumes have an essential role, since they represent a high proportion of diet. Forages commonly used in Mediterranean area can be a natural source of bioactive compounds that can be transferred to animal products. In order to evaluate and compare the potential contribution of some grass and legume species, to increase the level of bioactive compounds and antioxidant capacity in milk, Refs. [48, 57] compared the total polyphenol intake of three grasses, *Festuca arundinacea*, *H. vulgare* and *Triticosecale*, and four legumes, *Pisum sativum*, *Trifolium alexandrinum*, *V. sativa* and *Vicia faba minor*, given to seven groups of Mediterranean Red goats without supplement for eleven days. The single forage was cut daily and given *ad libitum* indoor. After an adaptation period, forage samples and milk samples of each group were collected and analysed for polyphenolic compounds and total antioxidant capacity. Folin-Ciocalteu method as described by Ref. [60] was used to determine tannic and non-tannic polyphenol contents in forage samples, after the addition of insoluble matrix polyvinylpolypyrrolidone (PVPP) and total and free polyphenol contents in milk samples. Contents were expressed as gallic acid equivalents (GAE). Milk conjugate polyphenol content was obtained by difference between milk total and free polyphenol contents. Total antioxidant capacity (TAC) was measured using the ferric-reducing antioxidant power as indicated by Benzie and Strain method and was expressed as μM FeSO<sup>4</sup> .

Among forages, *T. alexandrinum* and *F. arundinacea* were shown to enhance the milk-free polyphenol content (**Figure 9**) and the antioxidant capacity (**Figure 10**), respectively.

The phenological stage to which the fodder was used by goats may have contributed, as reported in the literature [47], to total polyphenol intake (**Figure 9**) and milk polyphenol content. As polyphenolic beneficial compounds occur largely in forages, it could be assumed their possible relationships and their transfer from diet, through some biotransformations or, as such, to milk according to De Feo et al.'s [14] study.

#### *2.2.4. Case study 4: Sulla forage and phenolic compounds and antioxidant capacity*

Among plant species that are used in ruminant feed in the Mediterranean area, Sulla (*Sulla coronarium* L.), which is a short-lived perennial legume, plays a key role in the cereal-based systems that are used in semiarid regions. This legume forage has widespread availability in Mediterranean areas, where it is greatly appreciated for the positive effects of its nutrient and CT contents on milk yield and composition, as demonstrated in both sheep and goats [58, 59].

**Figure 10.** Comparison of total antioxidant capacity (TAC) in milk from goats fed *ad libitum* with a single species in

HB = mixed hay plus 800 g/d of barley meal; SUL = Sulla fresh forage; SULB = Sulla fresh forage plus 800 g/d of barley

**Table 1.** Daily intake of polyphenol compounds, milk content and total antioxidant capacity according to feeding regimen

**HB SUL SULB**

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1.26<sup>b</sup> 3.72a 3.24a

**Feeding regimen**

Total polyphenol intake (g of GAE/d) 1.53b 9.20<sup>b</sup> 8.88<sup>a</sup>

Tannin intake (g of GAE/d) 0.26<sup>b</sup> 5.48<sup>a</sup> 5.64<sup>a</sup> Condensed tannin intake (g of DE/d) 3.5c 47.2a 35.6<sup>b</sup> Milk total polyphenols (g of GAE/d) 0.819<sup>b</sup> 0.964<sup>a</sup> 1.081<sup>a</sup> Milk-free polyphenols (μg/mL of GAE) 49.3<sup>b</sup> 56.7<sup>a</sup> 56.2<sup>a</sup> Total antioxidant capacity (log μmol/L) 2.38<sup>f</sup> 2.43e 2.47e

a–f Values within a row without a common superscript letter are significantly different (*P* ≤ 0.05).

pureness, given fresh (modified from Ref. [48]). a, b and c = *P* < 0.05.

meal. GAE = gallic acid equivalent. DE = delphinidin equivalent,

Non-tannic polyphenol intake (g of

GAE/d)

[60].

**Figure 9.** Comparison of total polyphenol intake (a) from goats fed *ad libitum* with a single species, in pureness, given fresh and milk total polyphenol content (b) from goats fed with the same forage species (modified from Ref. [48]). a, b, c, d, e, f = *P* < 0.05.

The results of a recent study [60] on three groups of *Girgentana* goats fed with Sulla fresh forage *ad libitum*, Sulla fresh forage *ad libitum* plus 800 g/d of barley meal and mixed hay *ad libitum* plus 800 g/d of barley meal indicate that Sulla fresh forage improves the plasma oxidative statuses of goats [61], milk total polyphenol (**Table 1**) content and the total antioxidant capacity of milk. Methods for milk total polyphenols and milk TAC assays are given in Section 2.2.3. Milk total polyphenol content seems closely related to its antioxidant activity. This fresh forage exerts antioxidant capacity due to its secondary compounds, which provide additional value in terms of oxidative status, and Sulla fresh forage seems to be a promising strategy for improving product quality.

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**Figure 10.** Comparison of total antioxidant capacity (TAC) in milk from goats fed *ad libitum* with a single species in pureness, given fresh (modified from Ref. [48]). a, b and c = *P* < 0.05.


HB = mixed hay plus 800 g/d of barley meal; SUL = Sulla fresh forage; SULB = Sulla fresh forage plus 800 g/d of barley meal. GAE = gallic acid equivalent. DE = delphinidin equivalent,

a–f Values within a row without a common superscript letter are significantly different (*P* ≤ 0.05).

The results of a recent study [60] on three groups of *Girgentana* goats fed with Sulla fresh forage *ad libitum*, Sulla fresh forage *ad libitum* plus 800 g/d of barley meal and mixed hay *ad libitum* plus 800 g/d of barley meal indicate that Sulla fresh forage improves the plasma oxidative statuses of goats [61], milk total polyphenol (**Table 1**) content and the total antioxidant capacity of milk. Methods for milk total polyphenols and milk TAC assays are given in Section 2.2.3. Milk total polyphenol content seems closely related to its antioxidant activity. This fresh forage exerts antioxidant capacity due to its secondary compounds, which provide additional value in terms of oxidative status, and Sulla fresh forage seems to be a promising strategy for improving

**Figure 9.** Comparison of total polyphenol intake (a) from goats fed *ad libitum* with a single species, in pureness, given fresh and milk total polyphenol content (b) from goats fed with the same forage species (modified from Ref. [48]). a, b,

product quality.

c, d, e, f = *P* < 0.05.

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**Table 1.** Daily intake of polyphenol compounds, milk content and total antioxidant capacity according to feeding regimen [60].

#### *2.2.5. Case study 5: degree of antioxidant protection*

In this case study, Ref. [13], in order to trace and identify milk and cheese from different feeding systems, proposed an interesting tool. Milk and cheese samples from ten feeding systems as grazing, grazing plus different types of supplement and indoor and zero grazing were studied to identify a tracing parameter correlated to the feeding system. In particular, α-tocopherol and cholesterol were measured in milk and cheese and were combined to calculate the degree of antioxidant protection (DAP). This tracing parameter was calculated as molar ratio between antioxidant compounds and a selected oxidation target. In dairy products from goats, only α-tocopherol was selected as the antioxidant because β-carotene is absent in goat's milk, and cholesterol was selected as oxidation target. All samples were analysed for α-tocopherol and cholesterol content. Briefly, all samples were hydrolysed in alkaline solution, and the extracted residue was dissolved in 2-propanol (1%) in n-hexane and analysed by the normal phase chromatographic method described in Ref. [13]. This index allows an evaluation of milk and cheese resistance to oxidative reactions, the main determinants of food quality and functionality for human nutrition. The DAP values (**Figure 11**) greater than 7.0 × 10−3 were found in grazing feeding systems, and values lower than 7.0 × 10−3 were found in indoor and zero grazing feeding systems, for milk and cheese.

These results show that cholesterol was highly protected against oxidative reactions when herbage was the only feed or was dominant in the goat diet. A strong positive correlation between herbage intake and DPA values allows to identify a linear regression: *y* = 0.12*x* + 5.52, where *y* = DPA (×10−3) and *x* = contribution of grazed herbage intake to the animal diet calculated as a percentage of the maximum intake of mature Maltese goats (1100 g/d = 100% grazing). The DAP index equal to 7.0 × 10−3 was able to distinguish dairy products when the grazed herbage in the goats' diet exceeded 15%. The reliability of DAP to measure the antioxidant protection of cholesterol appeared more effective when the feeding system was based on grazing than when cut herbage or zero grazing was utilised indoors by animals.

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**3. The role of the breed on oligosaccharides: a special focus on the** 

Besides the feeding system, the breed plays a fundamental role in affecting the nutritional profile of goat milk and cheese. The breed may be considered the result of the adaptation of a species to a given environment, basically in order to go over the climate and feeding and water resource limits that might affect the reproduction and kidding. The goats are present in high mountains as far as in the internal lands and coastal regions; they are reared in technological farms but also in extensive, grazing systems in the Mediterranean area, an environment characterised by high variability, that was able to select very different breeds [62].

The so-called native breed has become able to optimise the resources in terms of water and feedstuff [63]. The breed's answer is expressed as phenotype, quantity and, overall, quality of production. The differences are both in micro and macronutrients, and they are affected by the environment directly or indirectly. In the first case, we can say that different breed means different feeding behaviour and thus milk yield and quality, since it is well known that feeding largely affects the milk composition [64]. Moreover, the genetic polymorphism may affect

Within the same breed, in the same environment and diet, it is expectable to have very similar performances. Contrarily, especially for goat, significant differences have been found for quality but also quantity parameters. This variability has been explained, in part, by the genetic polymorphism of caseins that are αs2-casein, β-casein and k-casein but in particular at the locus αs1-casein, first discovered by Boulanger et al. [65]. It was found that goats carrying

phosphorus and smaller micelles than the milk from goats with weak alleles (FF) [66, 67]. Several goat breeds have been characterised for this variability: the Vallesana, Roccaverano, Jonica, Garganica and Maltese breeds [68] and Alpine breed [69]. Spanish Malagueña goats with a high (HG) and low (LG) genetic capability for αS1-casein synthesis were used to determine whether the different genotypes were related to differences in feed utilisation (13.6 vs. 17.7% crude protein content for diets 1 and 2, respectively). The findings have let to explain the differences in milk composition between the two genotype groups by the greater nitrogen and energy utilisation of HG vs. LG goats [70]. Moreover, the interaction genotype x feeding

system was studied (e.g., see Ref. [71] on Malagueña dairy goat breed).

casein present higher percentage of milk casein, fat, calcium,

**Mediterranean goats**

the milk features.

strong alleles (AA) for high α-s<sup>1</sup>

**Figure 11.** (a) Degree of antioxidant protection (DAP) of milk from goats fed with G = grazing, GBCM = grazing plus 0.6 kg/d mixed barley and chickpeas grain, GMBM = grazing plus 0.6 kg/d mixed corn and broad beans grain and HS = pasture hay *ad libitum* plus 0.6/kg/d of commercial concentrate. (b) DAP of *Caciotta* cheese from goats fed with G = grazing, GUC = grazing plus unlimited concentrate and HUC = hay plus unlimited concentrate. (c) DAP of *Caciotta* cheese from goats fed with GVC = grazing on valley pasture, GMC = grazing on mountain pasture and ZG = zero grazing (modified from Ref. [13]). a, b and c = *P* < 0.05.

These results show that cholesterol was highly protected against oxidative reactions when herbage was the only feed or was dominant in the goat diet. A strong positive correlation between herbage intake and DPA values allows to identify a linear regression: *y* = 0.12*x* + 5.52, where *y* = DPA (×10−3) and *x* = contribution of grazed herbage intake to the animal diet calculated as a percentage of the maximum intake of mature Maltese goats (1100 g/d = 100% grazing). The DAP index equal to 7.0 × 10−3 was able to distinguish dairy products when the grazed herbage in the goats' diet exceeded 15%. The reliability of DAP to measure the antioxidant protection of cholesterol appeared more effective when the feeding system was based on grazing than when cut herbage or zero grazing was utilised indoors by animals.
