INTRODUCTION
Olive production is very widespread over the world. In 2008, olive orchards occupied 10,839 thousand ha in the world with an increase of 2,888 thousand ha from 1998; Mediterranean countries represent 95% of the world’s surface area of olive orchards and 96% of the world’s production [1]. The use of pomace as feed for animals has been considered even if pomace production is seasonal and adequate preservation and storage techniques are required. Ensiling alone or mixed with other feeds or urea or treated with alkali could be considered the cheapest way to preserve pomace together with its inclusion in the farm diet [2]. Another interesting way to guarantee good preservation of the byproduct could be the drying process applied to olive pomaces [3] allowing, by choosing the best operative conditions, to preserve the phenolic composition and to reduce the oxidative processes of the lipid fraction. Virgin olive oil mechanical extraction from stoned olive pastes is a new approach to the processing generally applied to improve oil quality but, at the same time, can also be considered a method to reduce the lignin content in the stoned pomaces, thereby increasing thus their attractiveness as animal feed [3]. Stoned olive pomaces contain residual oil, with a fatty acid composition dominated by oleic acid and, at the same time, show high amounts of bio-active phenols such as secoiridoids and lignans. For those reasons, they have been considered for their potential application as important sources of monounsaturated fatty acids (MUFAs) and natural antioxidants in animal feeding [4].
The dried stoned olive pomaces, obtained as extravirgin olive oil mechanical extraction residue and processed as previously described [3], have been already used in the diet of dairy ewes [5] and for dairy water buffaloes [6]: in both experiments an improvement of the milk fatty acids profile and an improvement of the milk oxidative stability have been observed, furthermore a study [6] reported the occurrence of hydroxytyrosol in milk. The cultivation of olives in the Mediterranean basin often coincide with the breeding of sheep and buffaloes whose productions are for local people an economic resource [6,7]. In Italy the main product of buffalo is the milk which is almost entirely converted into mozzarella cheese. This is a soft cheese and it is very appreciated by consumers in Italy and other EU countries and in the USA.
The aim of this work on the basis of previous outcomes [6] is to verify the effect of further increasing the level of dried stoned olives pomaces (DSOP) in diet for lactating Mediterranean buffaloes, on the quantity and quality of milk produced and dietary-sensory characteristics of mozzarella cheese.
MATERIALS AND METHODS
Animals and diets
Animals
Sixteen pluriparous Mediterranean buffaloes (Bubalus bubalis, L.) were allotted into two groups, control (C) and treated (DSOP), with no significant differences in milk yield and lactation length in their previous lactation and no significant differences in days in milking, milk yield, live weight and body condition score (BCS) at beginning of the trial (Table 1). The experimental period lasted 40 days. The animals were weighed at the beginning and the end of the trial and the nutritional status was determined using the BCS, utilizing the scale modified [8] for buffalo; this method provides for the use of a score from 1 to 9. Buffalo’s cows were milked twice daily at intervals of 12 h using a milking machine and milk yields were measured at the on-set of the trial and after 15, 30, and 40 d after the beginning of the trial.
Diets
The diets were characterized by 26% 2nd cut alfalfa hay, 30% corn silage, and 44% concentrate for both groups, on a dry matter (DM) basis, and fed as a total mixed ration once a day. The formulation of concentrates and the chemical composition of the feedstuffs and of the two diets utilized are reported in supplementary material. The experimental concentrate contained 20.00% as fed of DSOP (Supplementary Table S1), dried according to the procedure proposed by other authors [3]. The control and experimental concentrates had a net energy content of 1.06 and 1.04 milk forage unit (FU)/kg DM and crude protein (CP) content of 192.0 and 190.2 g/kg DM respectively. The control and experimental diets were isoenergetic and isoproteinc, the control diet had 0.86 milk FU/kg DM and 149.5 g/kg DM of CP, experimental diet had 0.87 milk FU/kg DM and 148.7 g/kg DM of CP (Supplementary Table S2); each animal of two groups received 17 kg DM/d.
Each animal in the experimental group received daily 1.694 kg of stoned pomace corresponding to 1.602 kg of DM which accounted for 9.42% of the total DM of the diet.
Animals were kept, maintained and treated in accordance with International Guiding Principles for Biomedical Research Involving Animals that are accepted standards for animal welfare.
Feed sampling and analysis
Samples of forages, concentrates and dried stoned olive pomace were weekly collected and submitted for the duplicate chemical analyses.
The DM, CP, crude fibre (CF), ether extract (EE), and ash [9] as well as neutral detergent fibre, acid detergent fibre, and acid detergent lignin were determined [10]. In addition, the non-structural carbohydrates were calculated. The net energy of feeds, expressed as milk FU/kg DM, was calculated by using the chemical composition and the digestibility of the organic matter. The above listed parameters for both diets were calculated proportionally to the percentage of the feedstuffs components.
Milk sampling and analyses
At each control, individual milk samples from the morning and evening milking were collected and allotted into two aliquots for analysis. The first aliquot was processed to assess fat, protein (N× 6.38), lactose, urea and pH [11]; the second aliquot was processed to assess the milk coagulating parameters: rennet clotting time (r), curd firming time (K20), curd firmness (A30), estimated by means of the thromboelastograph Formagraph as previously reported [12] at 35°C using a liquid rennet (1:15,000 rennet unit), 90% chymosin diluted 1:100 and added to whole milk as 200 μL/100 mL.
The daily production of mozzarella cheese was estimated [13].
Mozzarella cheese and governing liquid sampling, analysis and oxidative stability evaluation
All the bulk milk of each group, the last day of the test, was processed according to the EU rules [14] to produce mozzarella cheese. The mozzarella cheese and governing liquid samples were collected according to the following procedure: mozzarella cheese was divided into two halves and was subsequently homogenized with an Omnimixer homogenizer at low speed without other solutions; the governing liquid was filtered with Whatman No. 1 filters. Mozzarella cheese samples were collected and allotted into three aliquots. The first aliquot was immediately processed for the following duplicate analytical determinations: fat, protein, ash and pH according to the National official methods [15]. The second aliquot was stored at −80°C until fatty acids composition which was determined by gas chromatographic as the methyl ester derivatives after trans-esterification with sulphuric acid following the procedure reported in a former study [6]; to evaluate the risk for coronary disease, atherogenic and thrombogenic indexes were calculated [16]. The third aliquot was stored at −80°C until analysis to assess carotenoids and tocopherols [17].
To assess the oxidative stability of mozzarella cheese and governing liquid, three mozzarella cheese samples and governing liquid were collected immediately and after a conservation period of 4 days (96 hours) at 4°C and darkness, and then stored at −80°C, until duplicate analytical determinations. The oxidative stability of the mozzarella lipid fraction and governing liquid were measured by the dosage of thiobarbituric acid-reactive substances (TBARs) [18] and expressed in μg of malonyldialdheyde (MDA) per g of fat and μg of MDA per mL, respectively. The evolution of α-tocopherols in mozzarella cheese was also assessed [17].
Mozzarella cheese sensory evaluation
At the end of the trial, a sensory test was carried out by seven trained judges on mozzarella cheese obtained from the bulk of the milk produced by the two groups during the last day of experimentation, using a descriptive questionnaire [19].
Statistical analysis
All parameters except the tocopherols and oxidative status of mozzarella cheese and governing liquid oxidative status were evaluated using the PROC general linear model (GLM) of SAS [20] according to the following linear model:
Fixed effect: dietary treatment C and DSOP.
The tocopherols and oxidative status of mozzarella cheese and governing liquid oxidative status data were evaluated using the PROC GLM of SAS [20] according to the following linear model:
Fixed effects: dietary treatment (C and DSOP); storage time (0 and 96 hours).
Results collected from the sensory evaluation of mozzarella samples have been elaborated by the Fisher’s exact test using the PROC FREQ of SAS [20].
RESULTS AND DISCUSSION
Characteristics of stoned olive pomace
The chemical composition and the net energy of the DSOP used are reported in supplementary material (Supplementary Table S3). The DM (945.4 g/kg) was similar to that present in the literature [6] (956.7 g/kg). The chemical composition in terms of CP, CF, and EE (80.8, 325.6, and 135.4 g/kg DM) was similar to the data in the literature [21] for a stoned virgin pomace (92.0, 317.0, and 137.0 g/kg DM). The net energy (0.56 milk FU/kg DM) was similar to that of a mature oat hay or of a bloom tall fescue hay (both around 0.58 milk FU/kg DM) while the protein level was intermediate between those two kinds of hay (48.0 and 96.5 g/kg DM). The phenolic profile of the DSOP showed the occurrence of verbascoside and 3,4-Dihydroxyphenylethanol-elenolic acid di-aldehyde, an aglycon derivative of the oleuropein (3,4-DHPEA-EDA) as the most abundant phenolic compounds, other than p-Hydroxyphenylethanol-elenolic acid di-aldehyde (p-HPEA-EDA), 3,4-DHPEA, p-HPEA, and (+)-1-acetoxypinoresinol. The total amount of phenolic compounds was 26.7 g/kg of DM.
Animal performance and milk characteristics
Animal performances
Table 2 shows the DM intake, milk production, its quality, the live weight, the average daily gain and BCS. The amount of DM ingested (17.00 kg/d), at this stage of lactation, is the maximum capacity of ingestion for the buffalo species [22]. The DSOP administered in the experimental group reached 1.602 kg DM/head/d, representing the 9.42% of the total DM of the experimental diet, higher than that found in a previous experimentation with a similar diet composition [7], which accounted 6.17% (1.050 kg DM/head/d). This result is in agreement with a preceding study [23], where no negative influences on DM intake in dairy cows receiving around the 15% of DM of ensiled olive cake, were observed. Dietary treatment did not affect milk yield and milk composition, in fact by comparing milk yield from the two groups (8.42 and 8.53 kg/d in C and DSOP group, respectively) no significant differences were observed. Furthermore no significant differences in the average daily gain (406 and 393 g/d) and in the BCS (6.50 and 6.69) have been observed.
Milk characteristics
Fat, protein, lactose and urea content were 8.57%, 4.78%, 4.88%, 39.10 mg/100 mL in milk from C group and 8.95%, 4.65%, 4.57%, and 40.52 mg/100 mL in milk from DSOP group, without statistical differences between the two experimental groups. These results are consistent with literature [22] and provide further confirmation of the absence of a negative effect of long chain unsaturated fatty acids (UFA) provided by the DSOP on the activity of rumen bacteria [4,6]. There were no statistically significant differences between milk from C group and the milk from DSOP group in terms of pH (6.73 and 6.72), rennet clotting time (18.34 and 17.87 min), curd firming time (2.02 and 2.34 min), curd firmness (50.73 and 48.63 mm) and estimated yield of mozzarella cheese (2.23 and 2.25 kg/d). The results of the coagulation parameters were similar to those found by another investigation [22], and better, for milk of DSOP group, than those previously reported [6] for the lower value of rennet clotting time (r = 17.87 vs 22.35 min) and curd firming time (K20 = 2.34 vs 2.66 min) and for the highest value of curd firmness (A30 = 48.63 vs 35.25 mm). It could be considered that the higher level of dietary DSOP (9.42%) compared to the quantity administered (6.17%) in the aforementioned trial [6] leads to a milk with better attitude for cheese making; then adding in the diet the stoned pomace did not determine negative changes both in quantity and quality of milk.
Mozzarella cheese chemical composition
In Table 3 is reported the effect of DSOP on pH and chemical composition of buffalo “mozzarella cheese”. All the parameters for the samples from the two groups were similar with the exception of the fat which was significantly higher in the mozzarella produced using the experimental group milk (25.24% vs 26.06%, p<0.05). Other researchers [24] obtained an average value of 26.70% of fat, and others [25] observed a slightly higher value (28.46%), whilst the datum from the National food composition tables [26] is lower (24.40%). Protein and ashes in the control and experimental groups (14.18% and 14.04%, 1.39% and 1.31%) were within the range (11.90%÷16.57%, 1.06%÷1.51%) described in a previous paper [25], and the National tables [26] reported a protein concentration of 16.70%. Furthermore, also the results expressed on the basis of the DM are significantly different for the fat content (58.81% vs 59.93%, p<0.05), thus the amount of DSOP in the concentrate promoted this modification in the “mozzarella cheese”.
Mozzarella cheese fatty acid composition
Table 4 reports the fatty acid composition of the lipid fraction in the mozzarella obtained with milk from the control and the experimental group. The percentages of C6:0, C8:0, C10:0, and C14:0 were significantly higher in the “mozzarella cheese” from the control group milk. The C18:1n9 content, in the “mozzarella cheese” from the milk of treated animals was higher (14.06% vs 20.90%, p<0.01); the oleic acid content of milk and consequently of “mozzarella cheese” could depend directly on the feed source and also on the mammary gland desaturation activity [27]. Among saturated fatty acids (SFA), C12:0, C14:0, and C16:0 are known to be able to increase the level of low density lipoprotein (LDL) cholesterol in the blood and in the serum with an aggravation of the coronary heart disease (CHD) risk in humans. On the contrary the augmentation of the consumption of dietary sources of MUFA and polyunsaturated fatty acids (PUFA) reduces the risk of the occurrence of CHD by lowering the level of LDL cholesterol in serum. Moreover MUFA are less sensitive to peroxidation respect to PUFA, thus their higher relative occurrence leads to a longer shelf-life in food products, with a slowdown in the onset of rancidity. A lower level of SFA (77.69% vs 71.63%, p<0.01), a higher UFA and MUFA content (22.31% vs 28.37%, p<0.01; 18.77% vs 24.65%, p<0.01), a lower n6/n3 ratio (3.76 vs 2.73, p<0.05) were found in mozzarella from buffalo cows fed with stoned pomace. Consequently lower atherogenic (4.95 vs 3.68, p<0.01) and thrombogenic (3.68 vs 3.14, p<0.05) indexes values were found in mozzarella cheese from DSOP group. These results put in evidence a strong improvement of the healthy value of “mozzarella cheese” produced by milk from DSOP group. In fact, the utilization of diets characterized by low values of these indices is correlated with a reduction of the potential risk of CHD [28].
Mozzarella cheese tocopherols and oxidative status
The effects of diet and storage on “mozzarella cheese” tocopherol content and oxidative status of cheese and governing liquid are shown in Table 5. The total tocopherols’ concentrations found in the mozzarella samples from the control group and experimental group was 161.27 and 149.90 μg/100 g respectively, similar to those found in literature [29] in commercial mozzarella cheeses produced from water buffalo milk. However no statistical differences due to dietary treatment were found in the concentration of tocopherols. The storage period from 0 to 96 hours resulted in a significant reduction (p<0.01) in the α-tocopherol content, which was slight higher in mozzarella from the control group (Figure 1). In dairy products, tocopherols are inhibitors of the free radical action that catalyze the beginning and propagation of chain reactions in lipid peroxidation [30]. No significant differences were observed between the TBARs according to the two groups (2.69 and 2.57 μg MDA/g fat) and Figure 2 put in evidence the consumption of antioxidants such as tocopherols during storage. Also in the governing liquid, the differences found for the two groups were not significant (0.38 and 0.26 μg MDA/mL). Nevertheless, the slightly lower level of MDA in modulus, in the governing liquid of mozzarella obtained from treated animals could be related to a partial transfer of hydrophilic polyphenols, with antioxidant activity, from cheese to liquid. Furthermore, also in the governing liquid during the storage period a consumption of antioxidants such as tocopherols was observed (Figure 3).
Sensory evaluation of mozzarella cheese
In Table 6, the results of the sensory evaluation of the mozzarella cheese samples obtained from the bulk milk of the two groups are reported as number of attributions per descriptor. No statistical differences were observed between the two samples for all descriptors using the Fisher’s exact test. Most of the judges have indicated for the surface a pearly white color and a smooth rind; the internal aspect has been described as pearly white and with good compactness. For both kind of mozzarella samples, the described olfactory sensations were mostly lactic and the prevalent taste sensation was sweet. Texture resulted mainly soft and juicy, in particular in mozzarella from the DSOP fed group. As a general consideration, the two mozzarella types received a similar and positive sensorial evaluation confirming the inclusion of the virgin stoned pomace in the experimental diet not to cause any negative effect not even in the sensory properties of the mozzarella cheese.
IMPLICATIONS
The results from this study have confirmed that the inclusion of a dried stoned olive pomace, at the tested concentration in dairy water buffalo feed does not provoke any damage or significant performance worsening without reduction in the quantity and quality of milk produced. On the contrary, important effects on mozzarella cheese quality were shown; the dried stoned olive pomace supply led to a modification of fat content and the fatty acid profile with an increment in the MUFAs. Further, the dietetic-nutritional characteristics of the mozzarella cheese are improved due to a better fatty acid composition determined by a decrease in the saturated/unsaturated ratio and of the atherogenic and thrombogenic indices.
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