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The inclusion of fresh forage in the lactating buffalo diet affects fatty acid and sensory profile of mozzarella cheese

Open ArchivePublished:May 23, 2018DOI:https://doi.org/10.3168/jds.2018-14710

      ABSTRACT

      The aim of this study was to determine the effect of inclusion of fresh forage in diet for lactating buffalo on properties of mozzarella cheese under intensive farming conditions. Thirty-two buffalo cows were equally allotted into 2 groups fed diets with (fresh group, FRS) or without (control group, CTL) fresh sorghum. The study consisted of 2 trials. In the first one, animals from group FRS were fed a diet containing 10 kg of fresh sorghum (10-FRS diet) that was doubled to 20 kg (20-FRS diet) in the second trial. All diets were isonitrogenous and isoenergetic, and fresh forage accounted for 13.4 and 26.5 of dietary dry matter, respectively, for the 10-FRS and 20-FRS diet. In each trial, milk from the 2 groups was used to produce 3 batches/diet of Mozzarella di Bufala Campana Protected Designation of Origin cheese. Milk yield and composition were not influenced by dietary treatment. The use of 10-FRS diet did not affect any properties of mozzarella. As the inclusion rate of fresh sorghum doubled to 20 kg, an increment of unsaturated fatty acid percentages and a lowering of short-chain and saturated fatty acids were observed. Moreover, the sensory characteristics of mozzarella were modified, although no effects were observed on consumer acceptance. We conclude that the use of green fodder can represent a low-cost feeding strategy to improve the healthiness of buffalo mozzarella under intensive farming conditions with no detrimental effect on consumer blind acceptance.

      Key words

      INTRODUCTION

      Dairy water buffalo (Bubalus bubalis) farming is a traditional Italian enterprise that in recent years has been involved in intensification of rearing techniques (
      • Napolitano F.
      • De Rosa G.
      • Grasso F.
      • Pacelli C.
      • Bordi A.
      Influence of space allowance on the welfare of weaned buffalo (Bubalus bubalis) calves.
      ). Buffalo milk is almost exclusively used for cheese-making mozzarella (
      • Masucci F.
      • De Rosa G.
      • Barone C.M.A.
      • Napolitano F.
      • Grasso F.
      • Uzun P.
      • Di Francia A.
      Effect of group size and maize silage dietary levels on behaviour, health, carcass and meat quality of Mediterranean buffaloes.
      ), a typical fresh and stringy-textured cheese, that has been endowed (EC 103/2008) with Protected Designation of Origin (PDO) Mozzarella di Bufala Campana. In the last few years, an increasingly number of buffalo farms have spread outside the PDO area to take advantage of the high price paid for buffalo milk and to differentiate dairy products (
      • Cecchinato A.
      • Penasa M.
      • Cipolat Gotet C.
      • De Marchi M.
      • Bittante G.
      Short communication: Factors affecting coagulation properties of Mediterranean buffalo milk.
      ). In such increasingly competitive market, buffalo dairy farmers producing mozzarella-PDO are forced to pursue competitive strategies focusing on product quality. The increasing consumer interest in nutritional and health properties of foods could create new market opportunities (
      • Jones P.J.
      • Jew S.
      Functional food development: Concept to reality.
      ;
      • Siró I.
      • Kápolna E.
      • Kápolna B.
      • Lugasi A.
      Functional food. Product development, marketing and consumer acceptance—A review.
      ;
      • Annunziata A.
      • Vecchio R.
      Functional foods development in the European market: A consumer perspective.
      ).
      Dietary recommendations for human health indicate a reduction of SFA and trans fatty acids to reduce incidence of cardiovascular disease (
      • Kliem K.E.
      • Shingfield K.J.
      Manipulation of milk fatty acid composition in lactating cows: Opportunities and challenges.
      ). Depending on breed, diet, and stage of lactation, fat of milk and dairy products has SFA content over 60%, but it contains the health-promoting rumenic acid (cis-9,trans-11 C18:2, commonly referred as CLA), a naturally occurring anticarcinogen (
      • Jensen R.G.
      The composition of bovine milk lipids: January 1995 to December 2000.
      ). Therefore, interest is growing in the development of dairy products naturally enriched in PUFA and CLA. Several feeding strategies are known to be able to provide higher nutritional characteristics to milk fat (
      • Chilliard Y.
      • Ferlay A.
      Dietary lipids and forages interactions on cow and goat milk fatty acid composition and sensory properties.
      ;
      • Elgersma A.
      • Tamminga S.
      • Ellen G.
      Modifying milk composition through forage.
      ). In particular, the use of fresh forage may represent a low-cost approach in comparison with diet supplementation with oilseeds or fats and does not result in significant increases in trans 18:1 isomers other than trans-11 18:1 (
      • Dewhurst R.J.
      • Shingfield K.J.
      • Lee M.R.F.
      • Scollan N.D.
      Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems.
      ). In addition, consumers commonly prefer “green image” products obtained from grazing animals or, at least, fed without the use of preserved fodders (
      • Kalač P.
      The effects of silage feeding on some sensory and health attributes of cow's milk: A review.
      ).
      However, feeding management and fat characteristics may also affect the sensory quality of the dairy products (
      • Coulon J.B.
      • Delacroix-Buchet A.
      • Martin B.
      • Pirisi A.
      Relationships between ruminant management and sensory characteristics of cheeses: A review.
      ;
      • Dewhurst R.J.
      • Shingfield K.J.
      • Lee M.R.F.
      • Scollan N.D.
      Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems.
      ), whereas any perceived reduction of typical characteristics of a traditional food might be not accepted by regular consumers (
      • Vecchio R.
      • Lombardi A.
      • Cembalo L.
      • Caracciolo F.
      • Cicia G.
      • Masucci F.
      • Di Francia A.
      Consumers’ willingness to pay and drivers of motivation to consume omega-3 enriched mozzarella cheese.
      ).
      Total mixed rations based on maize and grass silages, hays, and concentrates are commonly used in buffalo farming throughout the year. The hypothesis is that fresh-cut forage inclusion in the diet for lactating buffalo would be able to improve the healthy characteristics of milk fat under intensive farming conditions. We used sorghum, a forage crop that is spreading in intensive dairy farming, due to its higher flexibility (compared with maize silage, it can be used both fresh and ensiled), and lower environmental impact (compared with maize silage it needs lower inputs of water and nitrogen fertilizer;
      • Lemaire G.
      • Charrier X.
      • Hébert Y.
      Nitrogen uptake capacities of maize and sorghum crops in different nitrogen and water supply conditions.
      ;
      • Farré I.
      • Faci J.M.
      Comparative response of maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) to deficit irrigation in a Mediterranean environment.
      ). Therefore, this study aimed to evaluate fatty acid profile, sensory properties, color, and consumer liking of Mozzarella di Bufala Campana PDO cheese produced under the dietary use of fresh sorghum.

      MATERIALS AND METHODS

      Experimental Design, Animals, Diets, and Cheese Production

      The study consists of 2 trials, 17 d each, and took place in September 2014 in a buffalo dairy farm (40°31′N 14°57′E, Campania region, southern Italy) producing PDO mozzarella cheese. Thirty-two lactating buffalo were blocked by milk yield and DIM and randomly allocated into 2 groups fed diets with (fresh group, FRS) or without (control group, CTL) daily cut fresh sorghum. Two inclusion rates of fresh sorghum (10 and 20 kg as fed) were tested. The lower rate was chosen in order not to markedly change the daily feeding routine and also to extend the period of fresh forage availability. However, this lower rate was unable to significantly change mozzarella fatty acid profile; therefore, it was doubled. The cows were housed into 2 adjacent freestall barns with access to the outdoors and were milked twice daily (0500 and 1700 h).
      In the first trial, the CTL group was fed the standard diet used by the farmer, whereas the FRS group was fed a TMR containing 10 kg of fresh sorghum (10-FRS diet). Fresh forage accounted for about 13.4% of TMR on a DM basis. The CTL and 10-FRS diets were formulated to be isonitrogenous and isoenergetic and were based on the same ingredients except for inclusion of fresh forage (Table 1). Sorghum [Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf.; commercial hybrid Nicol, Pioneer Hi-Bred International, Johnston, IA] was sown on the farm in July 2014 and at start of the trial was at the early milk stage [i.e., growth stage 5 to 6 according to the scale of
      • Vanderlip R.L.
      • Reeves H.E.
      Growth stages of sorghum [Sorghum bicolor, (L.) Moench.].
      ]. Sorghum was cut daily about 3 cm long and was mixed into the TMR with the other ingredients. The rations were fed once daily (0800 h) for ad libitum intake (approximately 10% orts) and were re-approached twice daily to ensure unlimited access to feed. The experimental period consist of 14 d of adaptation to diet and 3 d of cheese manufacturing. In each of them, daily (sum of pm and am milkings) bulk milk of each group was collected along with sampling of fresh sorghum, TMR, and milk of each cow. Group milk was transported to the dairy in refrigerated stainless-steel tanks and used for separately manufacturing mozzarella cheese according to the traditional procedure. Briefly, raw water buffalo milk was gently heated (37–38°C) and added with natural whey starter culture from the previous day manufacture and liquid rennet (Caglificio Clerici S.p.a., Codrago, Como, Italy). At curd formation, the coagulum was reduced to particles of 2 to 3 cm and held under whey until pH 4.85, a value suitable for manual stretching into hot water (90–95°C). Thereafter, the stretched curd was mechanically formed into 50-g small balls that were placed in brine (2% NaCl) and sent to the laboratory. A total of 3 batches/diet were produced, about 20 kg each. Over the 3 d of cheese manufacturing, yield (%) of mozzarella was 27.2 ± 0.21 and 27.1 ± 0.26, and DMI (kg/d) was 18.2 ± 1.15 and 17.9 ± 0.65 for CTL and 10-FRS groups, respectively.
      Table 1Ingredients and chemical composition of fresh sorghum (n = 2) and of the TMR fed to buffalo cows
      ItemFresh sorghumCTL TMR
      Total mixed ration containing no fresh sorghum.
      10-FRS TMR
      Total mixed ration containing 10 kg of fresh sorghum.
      20-FRS TMR
      Total mixed ration containing 20 kg of fresh sorghum.
      Ingredient, kg as fed
       Fresh-cut sorghum1020
       Maize silage181815
       Grass silage107.04.0
       Meadow hay4.53.02.5
       Maize meal1.52.03.2
       Ground corn silage2.51.5
       Concentrate
      Based on soybean meal, sunflower meal, and barley meal.
      2.83.33.1
       Mineral and vitamin mix0.250.250.25
       DM, kg17.618.018.2
       Forage % DM686969
      Chemical composition
       DM, %23.7
       NEL, MJ/kg of DM4.765.946.005.95
       Ether extract, % of DM2.52.82.93.0
       CP, % of DM7.114.513.613.7
       NDF, % of DM45.742.239.538.8
       ADF, % of DM34.928.028.529.1
       Starch, % of DM2.9120.219.818.1
      1 Total mixed ration containing no fresh sorghum.
      2 Total mixed ration containing 10 kg of fresh sorghum.
      3 Total mixed ration containing 20 kg of fresh sorghum.
      4 Based on soybean meal, sunflower meal, and barley meal.
      The second trial started immediately after the end of the first one. Other 32 animals were used and randomly allocated to the CTL and FRS-20 groups. The TMR for control group was kept constant, whereas group FRS was fed a diet in which the fresh sorghum content was doubled to 20 kg (20-FRS diet) and accounted for about 26.5% of DM. The 20-FRS diet was kept isonitrogenous and isoenergetic with respect to CTL (Table 1). Fresh sorghum was at the soft dough stage [i.e., growth stage 7 (
      • Vanderlip R.L.
      • Reeves H.E.
      Growth stages of sorghum [Sorghum bicolor, (L.) Moench.].
      )]. The same experimental design and sampling procedure reported above for the first trial were used. Average percent mozzarella yields were 28.6 ± 0.59 and 28.6 ± 0.80, whereas DMI (kg/d) were 19.1 ± 0.8 and 18.9 ± 0.9 for groups CTL and 20-FRS, respectively.

      Chemical Analyses of Milk, Feeds, and Cheese, and Instrumental Measures of Color and Texture of Cheese

      The samples of fresh sorghum and TMR collected over the 3 d of mozzarella manufacturing were pooled by type and analyzed for DM (air-dried oven at 65°C until constant weight), CP (Kjeldahl method), ash and ether extract (
      AOAC International
      Official Methods of Analysis.
      ), and NDF and ADF (
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and non starch polysaccharides in relation to animal nutrition.
      ). The NEL of the diets was calculated according to
      • Sauvant D.
      • Nozière P.
      La quantification des principaux phénomènes digestifs chez les ruminants: les relations utilisées pour rénover les systèmes d'unités d'alimentation énergétique et protéique.
      .
      Milk samples were analyzed the same day of collection for fat, protein, and lactose (MilkoScan FT 6000, Foss Electric, Hillerød, Denmark). Each batch of mozzarella (6 for CTL, 3 for 10-FRS diet, and 3 for 20-FRS diet) was separately analyzed the day after manufacturing. Overnight, the samples were stored at 10°C and were allowed to equilibrate at room temperature (22–23°C) before analysis. The color, chemical composition, and fatty acid composition were determined on 3 samples/batch about 200 g each. For each sample, the color was determined in quadruplicate and was separately measured in the inner and outer surface of cheese according the CIELAB system (spectrophotometer U-3000, Hitachi, Tokyo, Japan). Chemical and fatty acid compositions of each sample were determined in triplicate after grinding the samples. Moisture was determined by oven drying; fat and protein were quantified by the Gerber and Kjeldahl methods, respectively (
      AOAC International
      Official Methods of Analysis.
      ). Extraction of fat for fatty acid composition was carried out according the Schmidt–Bondzynski–Ratzlaff method with modifications as described by
      • Romano R.
      • Giordano A.
      • Chianese L.
      • Addeo F.
      • Spagna Musso S.
      Triacylglycerols, fatty acids and conjugated linoleic acids in Italian Mozzarella di Bufala Campana cheese.
      . The GC analysis was performed on a DANI Master gas chromatograph (Dani Instrument SPA, Cologno Monzese, Milan, Italy) instrument equipped with a Quadrex Bonded Cyanopropyl silicone capillary column (length 60 m, internal diameter 0.25 mm, film thickness 0.25 µm) according to the procedure outlined elsewhere (
      • Esposito G.
      • Masucci F.
      • Napolitano F.
      • Braghieri A.
      • Romano R.
      • Manzo N.
      • Di Francia A.
      Fatty acid and sensory profiles of Caciocavallo cheese as affected by management system.
      ). Fatty acid peaks in chromatograms were identified using the Supelco 37 Component FAME MIX (Supelco, Bellefonte, PA). Standards for CLA (C18:2 cis-9,trans-11) and trans vaccenic acid (C18:1 trans-11) were obtained from NuChek Prep (Elysian, MN). Values of individual fatty acids <0.1 were not quantified. Fatty acids were expressed as a percentage of total methylated fatty acids. Atherogenic index was calculated according to
      • Ulbricht T.L.V.
      • Southgate D.A.T.
      Coronary heart disease: Seven dietary factors.
      .

      Sensory Analyses of Mozzarella Cheese

      A panel consisting of 10 panelists (6 females and 4 males) with a mean age of 34 yr was used to perform 2 separate quantitative descriptive sensory analyses of the products obtained in each trial. Panelists were recruited and selected following the international standard ISO 8586–1 (
      ISO. (International Organization for Standardization)
      Sensory analysis–General guidelines for the selection, training and monitoring of selected assessors and expert sensory assessors.
      ) by assessing their capacity to identify the 4 basic tastes (sourness, sweetness, bitterness, and saltiness), as indicated by
      • Albenzio M.
      • Santillo A.
      • Caroprese M.
      • Braghieri A.
      • Sevi A.
      • Napolitano F.
      Composition and sensory profiling of probiotic Scamorza ewe milk cheese.
      . Then, panelists were trained on the use of the scale (
      • Stone H.
      • Sidel J.L.
      Sensory Evaluation Practices.
      ). Based on the available literature (
      • Muir D.D.
      • Hunter E.A.
      • Banks J.M.
      • Horne D.S.
      Sensory properties of hard cheese: Identification of key attributes.
      ,
      • Muir D.D.
      • Banks J.M.
      • Hunter E.A.
      Sensory properties of cheddar cheese: Effect of starter type and adjunct.
      ;
      • Murray J.M.
      • Delahunty C.M.
      Mapping consumer preference for the sensory and packaging attributes of Cheddar cheese.
      ;
      • Adhikari K.
      • Heymann H.
      • Huff H.E.
      Textural characteristics of low fat, full fat and smoked cheeses: Sensory and instrumental approaches.
      ), panelists developed a specific vocabulary for Mozzarella cheese and agreed on a 19-attribute consensus list (Table 2) concerning appearance, odor/flavor, taste, and texture (3, 6, 3, and 7 attributes were identified, respectively). Three points of the scale (low, medium, and high intensity) were anchored to the reference material during the panel training to build a specific reference frame for assessor training (Table 2). The panel leader guided the assessors in selecting the most appropriate references for at least 2 anchor points of each sensory attribute (
      • Albenzio M.
      • Santillo A.
      • Caroprese M.
      • Braghieri A.
      • Sevi A.
      • Napolitano F.
      Composition and sensory profiling of probiotic Scamorza ewe milk cheese.
      ). A quantitative descriptive analysis was used to evaluate the products (
      • Lawless H.T.
      • Heymann H.
      Data Relationships and Multivariate Applications. Food Science Text Series.
      ) obtained in the first trial: 10-CTL and 10-FRS. Tests started at 1030 h and were conducted in sensory booths (ISO 8589;
      ISO. (International Organization for Standardization)
      Sensory analysis–General guidance for the design of test rooms. Standard number ISO 8589.
      ). For the evaluation of appearance, booths were illuminated with white fluorescent lighting, whereas odor/flavor, taste, and texture attributes were assessed under red fluorescent lights to minimize color differences among samples. Samples (15–20 g cubes) were coded, randomized across panelists, replications, and samples, and served at 18°C. The intensity of each attribute was rated on 100 mm unstructured lines anchored at each end with 0 at the left end (attribute not perceived) and 100 at the right end (attribute perceived as very strong). Panelists drank a sip of water after each sample to make the conditions similar for each tasting. The interval between consecutive samples was roughly 10 min. The panelists received no information concerning the products under test and evaluated 2 replications of each product in one session. The same trained panel was used for the quantitative descriptive analysis of the products obtained in second trial (20-CTL and 20-FRS) as described in the previous one.
      Table 2Definition of the descriptive attributes used to assess mozzarella cheese
      DescriptorDefinition
      Appearance
       ColorOverall intensity of color (from white to ivory)
       BrightnessOverall intensity of the light reflected from the external surface
       SmoothnessOverall uniformity of the external surface
      Odor/flavor
       Overall odorOverall intensity of the odor
       Overall flavorOverall intensity of the flavor
       MilkOdor/flavor arising from milk at room temperature
       ButterOdor/flavor arising from butter at room temperature
       WheyOdor/flavor associated with whey
       YogurtOdor/flavor associated with plain whole yogurt
      Taste
       SaltyFundamental taste associated with sodium chloride
       SourFundamental taste associated with citric acid
       SweetFundamental taste associated with sucrose
      Texture
       TendernessMinimum force required to chew mozzarella samples: the lower the force, the higher the tenderness
       ElasticityDegree to which the original shape of a product is restored after compression between the teeth
       JuicinessMoisture released during mastication (low: saliva is absorbed by the product; high: liquids are abundantly released during mastication)
       CohesivenessThe degree to which a mozzarella sample holds together or adheres to itself while being chewed
       ChewinessEasiness to masticate the sample to a state pending swallowing
       ScreechyFriction of the product against the teeth, typical of milk casein soon after hot water stretching
      Consumer liking for mozzarella cheese was assessed only on the samples from the second trial using 94 untrained consumers (49 female and 45 male subjects) with an age ranging from 24 to 60 yr. Each consumer assessed two 15–20-g samples in random order in a controlled sensory analysis laboratory and in blind conditions. Participants were asked to express their overall liking for the 2 products. In addition, they were asked to express their liking for 3 specific aspects: texture, appearance, and taste/flavor. Participants used a 9-point hedonic scale with a central point corresponding to “neither pleasant nor unpleasant” (score = 5), a left end (score = 1) labeled as “extremely unpleasant” and a right end (score = 9) labeled as “extremely pleasant” (
      • Kähkönen P.
      • Tuorila H.
      • Rita H.
      How information enhances acceptability of a low-fat spread.
      ).

      Statistical Analysis

      Data from the 2 trials were separately analyzed by SAS, version 8.1 (SAS Institute Inc., Cary, NC). Two-way ANOVA per repeated measures (Mixed procedure) was used to test the effect of diet on milk yield and composition with treatment as nonrepeated factor and day of sampling as repeated factor. The cow variance was considered as random and used as the error term to test the main effect of diet. One-way ANOVA (Mixed procedure) was used to analyze data on chemical and fatty acid composition of mozzarella cheese. The batch of production was used as the error term to test the main effect of diet. For the variable color, 2-way ANOVA was performed as the effect of area of measurement (i.e., internal and external) was also examined.
      Sensory profile data from each trial were separately subjected to a preliminary ANOVA to verify the reliability of the panel. In particular, the following fixed effects were assessed: diet (2 levels), replication (2 levels), and assessor (10 levels), and the corresponding first-order interactions. Subsequently, a mixed procedure was used to evaluate the fixed effect of diet (2 levels) using replication (2 levels) and assessor (10 levels) as random factors. A t-test was used to assess consumer likings expressed for the products obtained in the second trial. Statistical significance was declared at P < 0.05 and tendencies discussed at P < 0.10.

      RESULTS AND DISCUSSION

      Milk Production and Chemical Composition and Color of Mozzarella

      Table 3 shows milk yield and composition of the 2 CTL groups, and the corresponding 2 experimental groups fed the diets containing fresh sorghum at 13.4 (10-FRS) and 26.5% DM (20-FRS). No effects of diet were observed for any parameters. Similarly, no significant differences were observed between CTL compared with 10-FRS and 20-FRS mozzarella for chemical composition, even if fat content tended (P = 0.103) to be higher in 20-FRS than in CTL mozzarella (Table 4). No differences were observed between groups FRS and the corresponding control groups in terms of mozzarella instrumental color. This result was expected because the white color is a basic requirement of buffalo mozzarella related to the physiology of these animals (
      • Jana A.H.
      • Mandal P.K.
      Manufacturing and quality of Mozzarella cheese: A review.
      ). Differences were found for lightness, redness, and yellowness of internal and the external area of mozzarella (P < 0.001) due to their different texture (data not shown).
      Table 3Yield and composition (LSM ± SEM) of milk obtained from buffalo fed fresh sorghum (10-FRS and 20-FRS received 10 and 20 kg of fresh sorghum, respectively) and the corresponding control groups (10-CTL and 20-CTL, respectively) fed no fresh forages (n = 48)
      Item10-CTL10-FRSP-value20-CTL20-FRSP-value
      Yield, kg/animal per d9.2 ± 0.289.3 ± 0.280.6898.7 ± 0.269.1 ± 0.260.558
      Chemical composition, g/kg
       Fat88.1 ± 2.8087.6 ± 2.800.89590.6 ± 3.3394.1 ± 3.330.240
       Protein49.9 ± 0.8948.4 ± 0.890.26150.7 ± 1.1252.7 ± 1.120.202
       Lactose48.2 ± 0.4348.9 ± 0.430.32447.1 ± 0.7147.1 ± 0.710.975
      Table 4Chemical composition and color of mozzarella cheese (LSM ± SEM) obtained from buffalo fed fresh sorghum (10-FRS and 20-FRS received 10 and 20 kg of fresh sorghum, respectively) and the corresponding control groups (10-CTL and 20-CTL, respectively) fed no fresh forages (n = 9)
      Item10-CTL10-FRSP-value20-CTL20-FRSP-value
      Chemical composition, g/kg
       Moisture491.7 ± 0.90487.5 ± 0.900.755492.4 ± 1.11479.8 ± 1.110.470
       Fat276.0 ± 0.28278.8 ± 0.280.505276.2 ± 0.36287.0 ± 0.360.103
       Protein208.7 ± 0.43204.2 ± 0.430.518206.9 ± 0.67201.3 ± 0.670.587
      Color
       L (lightness)95.5 ± 0.6395.2 ± 0.630.74993.3 ± 2.0093.6 ± 2.000.931
       a* (red-green)−2.7 ± 0.14−2.3 ± 0.140.132−2.7 ± 0.21−2.5 ± 0.210.532
       b* (yellow-blue)8.6 ± 0.318.5 ± 0.310.7468.7 ± 0.477.8 ± 0.470.247
      Fatty acid profile of mozzarella cheese under the different diets is presented in Table 5. Fatty acid composition of CTL and 10-FRS mozzarella did not differ, with the exception of C6:0 and C16:1 (P < 0.05) being lower in 10-FRS. In the second trial, 20-FRS mozzarella had lower values of C4:0, C6:0, C8:0 (P < 0.001), and C10:0 (P < 0.05), and higher contents of C18:1n-9 cis, C18:3n-3, C18:1 trans-11, CLA cis-9,trans-11, C22:0 (P < 0.001), C18:0, and C20:0 (P < 0.05). A tendency (P ≤ 0.10) was also observed for C14:0 and C16:0 to be lower. As consequences, 20-FRS mozzarella presented higher levels of PUFA and MUFA, lower percentages of SFA, and a better value of atherogenic index (P < 0.001).
      Table 5Fatty acid composition (% of total fatty acids) of mozzarella cheese (LSM ± SEM) obtained from buffalo fed fresh sorghum (10-FRS and 20-FRS received 10 and 20 kg of fresh sorghum, respectively) and the corresponding control groups (10-CTL and 20-CTL, respectively) fed no fresh forages (n = 9)
      Fatty acid10-CTL10-FRSP-value20-CTL20-FRSP-value
      C4:04.41 ± 0.134.50 ± 0.130.6615.09 ± 0.102.65 ± 0.10<0.001
      C6:03.14 ± 0.082.82 ± 0.080.0483.00 ± 0.041.51 ± 0.04<0.001
      C8:01.30 ± 0.061.09 ± 0.060.0701.24 ± 0.020.91 ± 0.02<0.001
      C10:03.81 ± 0.193.78 ± 0.190.9273.34 ± 0.023.45 ± 0.020.023
      C12:05.65 ± 0.115.48 ± 0.110.3365.44 ± 0.055.33 ± 0.050.255
      C13:00.56 ± 0.060.46 ± 0.060.33480.155 ± 0.090.159 ± 0.090.770
      C14:014.90 ± 0.1415.30 ± 0.140.16614.69 ± 0.1114.26 ± 0.110.059
      C14:10.90 ± 0.070.94 ± 0.070.6741.27 ± 0.011.29 ± 0.010.676
      C15:02.73 ± 0.172.59 ± 0.170.6152.04 ± 0.062.16 ± 0.060.200
      C16:034.38 ± 0.3234.63 ± 0.320.60134.23 ± 0.1433.63 ± 0.140.070
      C16:13.1 ± 0.092.7 ± 0.090.0503.30 ± 0.043.23 ± 0.040.3037
      C17:00.89 ± 0.040.79 ± 0.040.1360.77 ± 0.070.70 ± 0.070.556
      C17:10.48 ± 0.040.47 ± 0.040.9250.29 ± 0.010.31 ± 0.010.672
      C18:07.61 ± 0.168.02 ± 0.160.1388.80 ± 0.119.87 ± 0.110.022
      C18:1n-9 cis13.23 ± 0.3813.44 ± 0.380.71713.60 ± 0.1416.40 ± 0.14<0.001
      C18:1 trans-110.31 ± 0.020.34 ± 0.020.4100.50 ± 0.020.77 ± 0.020.0004
      C18:2n-6 cis1.72 ± 0.151.73 ± 0.150.9761.43 ± 0.021.47 ± 0.020.369
      C18:3n-30.24 ± 0.030.20 ± 0.030.3130.26 ± 0.010.75 ± 0.01<0.001
      CLA cis-9,trans-110.34 ± 0.020.37 ± 0.020.4590.30 ± 0.020.74 ± 0.02<0.001
      C20:00.15 ± 0.010.20 ± 0.010.1820.16 ± 0.010.25 ± 0.010.002
      C22:00.12 ± 0.010.16 ± 0.010.1210.01 ± 0.070.02 ± 0.070.0007
      Saturated79.70 ± 0.3479.86 ± 0.340.80179.01 ± 0.1775.01 ± 0.17<0.001
      Unsaturated20.31 ± 0.3520.19 ± 0.350.82720.95 ± 0.1124.95 ± 0.11<0.001
      MUFA18.0 ± 0.4417.9 ± 0.440.868018.9 ± 0.1722.41 ± 0.17<0.001
      PUFA2.23 ± 0.172.29 ± 0.170.9772.00 ± 0.032.96 ± 0.03<0.001
      ATI
      Atherogenic index [C12:0 + (4 × C14:0) + C16:0]/UFA.
      4.95 ± 0.125.0 ± 0.120.56544.7 ± 0.053.84 ± 0.050.0005
      1 Atherogenic index [C12:0 + (4 × C14:0) + C16:0]/UFA.
      Overall, although the fatty acid profile in mozzarella cheese fat was not dramatically modified, feeding 20 kg of sorghum/animal (group 20-FRS) allowed to triple the contents of C18:3, and markedly increased the content of CLA and PUFA. It is well established that fat from grazing animals or fed high fresh forage diets have higher proportions of PUFA and particularly n-3 fatty acids, versus conventional cheese because forages are naturally rich sources of C18:3n-3 (
      • Ellis K.A.
      • Innocent G.
      • Grove-White D.
      • Cripps P.
      • McLean W.G.
      • Howard C.V.
      • Mihm M.
      Comparing the fatty acid composition of organic and conventional milk.
      ;
      • Lourenço M.
      • Van Ranst G.
      • Vlaeminck B.
      • De Smet S.
      • Fievez V.
      Influence of different dietary forages on the fatty acid composition of rumen digesta as well as ruminant meat and milk.
      ). Moreover, long-chain UFA can inhibit mammary gland synthesis of short-chain fatty acids in milk fat, leading to a reduction of SFA (
      • Grummer R.R.
      Effect of feed on the composition of milk fat.
      ). The higher intake of C18:3 can result in an increment of C18:1 trans-11 produced in the rumen by biohydrogenation (
      • Bauman D.E.
      • Barbano D.M.
      • Dwyer D.A.
      • Griinari J.M.
      Technical note: Production of butter with enhanced conjugated linoleic acid for use in biomedical studies with animal models.
      ) and, as a consequence, in an increment of CLA cis-9,trans-11 by the action of Δ9-desaturase (
      • Kay J.K.
      • Mackle T.R.
      • Auldist M.J.
      • Thomson N.A.
      • Bauman D.E.
      Endogenous Synthesis of cis-9, trans-11 conjugated linoleic acid in dairy cows fed fresh pasture.
      ). It has been also suggested that green grass, due to the high concentrations of soluble nitrogen, sugars, and soluble fiber, can enhance the growth of rumen bacteria producing CLA cis-9,trans-11 or blocking biohydrogenation of C18:1 trans-11 in the rumen, thus leading to its accumulation and availability for conversion to CLA cis-9,trans-11 in the mammary gland via Δ9-desaturase (
      • Kelly M.L.
      • Kolver E.S.
      • Bauman D.E.
      • Van Amburgh M.E.
      • Muller L.D.
      Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows.
      ;
      • Nudda A.
      • McGuire M.A.
      • Battacone G.
      • Pulina G.
      Seasonal variation in conjugated linoleic acid and vaccenic acid in milk fat of sheep and its transfer to cheese and ricotta.
      ). While numerous studies investigated fatty acid composition of bovine milk fat, literature on buffalo is still limited (
      • Varricchio M.L.
      • Di Francia A.
      • Masucci F.
      • Romano R.
      • Proto V.
      Fatty acid composition of Mediterranean buffalo milk fat.
      ;
      • Zotos A.
      • Bampidis V.A.
      Milk fat quality of Greek buffalo (Bubalus bubalis).
      ;
      • Pegolo S.
      • Stocco G.
      • Mele M.
      • Schiavon S.
      • Bittante G.
      • Cecchinato A.
      Factors affecting variations in the detailed fatty acid profile of Mediterranean buffalo milk determined by 2-dimensional gas chromatography.
      ). The use of flax seeds determined changes of buffalo fat similar but larger than those observed in the present study (
      • Santillo A.
      • Caroprese M.
      • Marino R.
      • Sevi A.
      • Albenzio M.
      Quality of buffalo milk as affected by dietary protein level and flaxseed supplementation.
      ). In this respect,
      • Dewhurst R.J.
      • Shingfield K.J.
      • Lee M.R.F.
      • Scollan N.D.
      Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems.
      indicate that fresh forages are less efficient at altering milk fatty acids than fats or concentrates. Accordingly, in this study the inclusion of 10 kg of fresh sorghum was unable to modify fatty acid composition of mozzarella, whereas in dairy cattle (
      • Couvreur S.
      • Hurtaud C.
      • Lopez C.
      • Delaby L.
      • Peyraud J.L.
      The linear relationship between the proportion of fresh grass in the cow diet, milk fatty acid composition, and butter properties.
      ) the inclusion of fresh forage at 30% of dietary DM determined results similar to those we observed for the 20-FRS diet. Nevertheless, whereas oilseed or fat supplementation tend to increase the cost of the feeding ration, fresh forage has the potential to lower the feeding costs (
      • Borreani G.
      • Coppa M.
      • Revello-Chion A.
      • Comino L.
      • Giaccone D.
      • Ferlay A.
      • Tabacco E.
      Effect of different feeding strategies in intensive dairy farming systems on milk fatty acid profiles, and implications on feeding costs in Italy.
      ). In addition, it may be conveniently used for quality-identified products, such as Mozzarella di Bufala PDO cheese, as for these products any change of raw materials are restricted or even prohibited, whereas the origin of forage may give the basis for the “terroir” notion (
      • Verdier-Metz I.
      • Martin B.
      • Pradel P.
      • Albouy H.
      • Hulin S.
      • Montel M.C.
      • Coulon J.B.
      Effect of grass-silage vs. hay diet on the characteristics of cheese: interactions with the cheese model.
      ). Moreover, fresh forage feeding may allow product differentiation (
      • Tempesta T.
      • Vecchiato D.
      An analysis of the territorial factors affecting milk purchase in Italy.
      ) and increase mozzarella liking as a consequence of the increased expectations induced by the modified process characteristics (
      • Napolitano F.
      • Girolami A.
      • Braghieri A.
      Consumer liking and willingness to pay for high welfare animal-based products.
      ). According to
      • Vecchio R.
      • Lombardi A.
      • Cembalo L.
      • Caracciolo F.
      • Cicia G.
      • Masucci F.
      • Di Francia A.
      Consumers’ willingness to pay and drivers of motivation to consume omega-3 enriched mozzarella cheese.
      , a large share of consumers would be interested in mozzarella PDO with a better fatty acid profile, but most of them expect these added-value products without substantial extra costs.

      Sensory Properties

      The fixed ANOVA separately conducted for each trial showed that in both cases the interactions of assessor × replication and assessor × diet were not significant. In addition, the 2 products did not change their sensory properties across replications as the interaction replication × diet was not significant. Therefore, a first relevant result of the present study was the development of a specific reference frame for the training of panelists to be used in the sensory analysis of mozzarella cheese. Second, we noted that the inclusion of 10 kg of fresh forage per animal was unable to change the sensory profile of mozzarella cheese, whereas when 20 kg of sorghum was used, most of the attributes were able to discriminate the product obtained by feeding the buffalo fed fresh forage from that obtained by feeding no fresh forage (Table 6). In particular, in the second trial the trained panel perceived the mozzarella 20-FRS to have a lower overall odor, overall flavor (P < 0.05), milk (P < 0.01), and whey odor/flavor (P < 0.05). Previous studies obtained controversial results in terms of odor/flavor with some attributes increasing when animals were kept on pasture, other attributes decreasing, and no effect on odor intensity (see
      • Coulon J.B.
      • Delacroix-Buchet A.
      • Martin B.
      • Pirisi A.
      Relationships between ruminant management and sensory characteristics of cheeses: A review.
      , for a review). More recently
      • Coppa M.
      • Ferlay A.
      • Monsallier F.
      • Verdier-Metz I.
      • Pradel P.
      • Didienne R.
      • Farruggia A.
      • Montel M.C.
      • Martin B.
      Milk fatty acid composition and cheese texture and appearance from cows fed hay or different grazing systems on upland pastures.
      recorded higher intensities of overall odor and aroma in Cantal cheese obtained from grazing cows as compared with the indoor control system, whereas
      • Agabriel C.
      • Martin B.
      • Sibra C.
      • Bonnefoy J.C.
      • Montel M.C.
      • Didienne R.
      • Hulin S.
      Effect of dairy production systems on the sensory characteristics of Cantal cheeses: A plant-scale study.
      noted a decrement of odor intensity as a consequence of the consumption of fresh forage in grazing cows. However, none of these authors included pasta filata cheese in their studies, and process-related factors could interact with the feeding regimen in affecting cheese quality. Conversely,
      • Esposito G.
      • Masucci F.
      • Napolitano F.
      • Braghieri A.
      • Romano R.
      • Manzo N.
      • Di Francia A.
      Fatty acid and sensory profiles of Caciocavallo cheese as affected by management system.
      found higher intensities of most odor/flavor attributes describing Caciocavallo (a semi-hard pasta filata cheese) obtained in winter when the cows were kept indoors and received hay and concentrate compared with the same cheese manufactured in spring when the animals were managed outdoors and pasture represented the primary feeding source. In addition, mozzarella is a fresh product consumed within few hours from the end of the production process, whereas in all the other studies cheese was always ripened for at least 3 mo and ripening can markedly affect cheese sensory profile (e.g.,
      • Coppa M.
      • Ferlay A.
      • Monsallier F.
      • Verdier-Metz I.
      • Pradel P.
      • Didienne R.
      • Farruggia A.
      • Montel M.C.
      • Martin B.
      Milk fatty acid composition and cheese texture and appearance from cows fed hay or different grazing systems on upland pastures.
      ). In particular, the higher content of UFA in cheese produced from grazing animals can affect the development of odor/flavor active compounds only after an adequate ripening (
      • Farruggia A.
      • Pomiès D.
      • Coppa M.
      • Ferlay A.
      • Verdier-Metz I.
      • Le Morvan A.
      • Bethier A.
      • Pompanon F.
      • Troquier O.
      • Martin B.
      Animal performances, pasture biodiversity and dairy product quality: How it works in contrasted mountain grazing systems.
      ), although some cheese flavor molecules may also originate from ruminal enzymatic degradation of certain UFA (
      • Coulon J.B.
      • Delacroix-Buchet A.
      • Martin B.
      • Pirisi A.
      Relationships between ruminant management and sensory characteristics of cheeses: A review.
      ). Nevertheless, other milk/cheese components (e.g., terpenes) should be investigated to identify molecules and biological processes responsible for odor/flavor differences induced by fresh forage feeding.
      Table 6Sensory profile of mozzarella cheeses (LSM ± SEM) obtained from buffalo fed fresh sorghum (10-FRS and 20-FRS received 10 and 20 kg of fresh sorghum, respectively) and the corresponding control groups (10-CTL and 20-CTL, respectively) fed no fresh forages
      Attribute10-CTL10-FRSP-value20-CTL20-FRSP-value
      Appearance
       Color60.35 ± 1.4557.95 ± 1.450.267974.15 ± 2.4374.95 ± 2.430.831
       Brightness65.70 ± 1.9467.45 ± 1.940.537465.30 ± 2.4372.15 ± 2.430.074
       Smoothness67.70 ± 0.8666.80 ± 0.860.476461.55 ± 2.6774.00 ± 2.670.008
      Odor/flavor
       Overall odor59.15 ± 2.3858.20 ± 2.380.783681.45 ± 1.7474.80 ± 1.740.022
       Overall flavor56.60 ± 2.0160.45 ± 2.010.205265.15 ± 2.0857.70 ± 2.080.030
       Milk55.05 ± 1.7254.20 ± 1.720.733570.05 ± 2.0559.25 ± 2.050.004
       Butter49.90 ± 2.6449.05 ± 2.640.824248.90 ± 3.1251.75 ± 3.120.533
       Whey45.25 ± 2.4342.80 ± 2.430.492724.60 ± 2.0417.45 ± 2.040.032
       Yogurt41.70 ± 2.7740.05 ± 2.770.682820.60 ± 1.7021.35 ± 1.700.762
      Taste
       Salty43.55 ± 1.6648.10 ± 1.660.082039.20 ± 3.1036.00 ± 3.100.483
       Sour42.30 ± 2.2841.95 ± 2.280.915833.10 ± 1.3816.80 ± 1.38<0.0001
       Sweet23.55 ± 1.3922.15 ± 1.390.492919.40 ± 1.1128.75 ± 1.11<0.0001
      Texture
       Tenderness67.10 ± 2.6567.20 ± 2.650.979255.35 ± 2.5778.70 ± 2.57<0.0001
       Elasticity58.85 ± 2.6063.95 ± 2.600.195268.65 ± 2.1758.80 ± 2.170.009
       Juiciness70.55 ± 1.7172.00 ± 1.710.561147.20 ± 2.0569.60 ± 2.05<0.0001
       Cohesiveness66.10 ± 1.8167.95 ± 1.810.486177.25 ± 2.0062.35 ± 2.000.0003
       Chewiness67.00 ± 2.0669.80 ± 2.060.358491.15 ± 2.8966.65 ± 2.89<0.0001
       Screechy61.35 ± 2.6264.00 ± 2.620.490977.90 ± 2.1363.20 ± 2.130.0007
      In agreement with previous studies on pasta filata cheese (
      • Carpino S.
      • Mallia S.
      • La Terra S.
      • Melilli C.
      • Licitra G.
      • Acree T.E.
      • Barbano D.M.
      • Van Soest P.J.
      Composition and aroma compounds of Ragusano cheese: Native pasture and total mixed rations.
      ;
      • Bonanno A.
      • Tornambè G.
      • Bellina V.
      • De Pasquale C.
      • Mazza F.
      • Maniaci G.
      • Di Grigoli A.
      Effect of farming system and cheese making technology on the physicochemical characteristics, fatty acid profile, and sensory properties of Caciocavallo Palermitano cheese.
      ;
      • Esposito G.
      • Masucci F.
      • Napolitano F.
      • Braghieri A.
      • Romano R.
      • Manzo N.
      • Di Francia A.
      Fatty acid and sensory profiles of Caciocavallo cheese as affected by management system.
      ), in terms of taste we observed an increased intensity of the attribute sweet and a decreased intensity of the attribute sour (P < 0.001) when fresh forage at 20 kg/animal was included in the diet as compared with the control products. Other authors observed similar results for the attribute sour (
      • Agabriel C.
      • Martin B.
      • Sibra C.
      • Bonnefoy J.C.
      • Montel M.C.
      • Didienne R.
      • Hulin S.
      Effect of dairy production systems on the sensory characteristics of Cantal cheeses: A plant-scale study.
      ;
      • Coulon J.B.
      • Delacroix-Buchet A.
      • Martin B.
      • Pirisi A.
      Relationships between ruminant management and sensory characteristics of cheeses: A review.
      ), whereas
      • Frétin M.
      • Ferlay A.
      • Verdier-Metz I.
      • Fournier F.
      • Montel M.-C.
      • Farruggia A.
      • Delbès C.
      • Martin B.
      The effects of low-input grazing systems and milk pasteurisation on the chemical composition, microbial communities, and sensory properties of uncooked pressed cheeses.
      found opposite results in uncooked cheese but only when pasteurized milk was used.
      The 2 experimental products were also discriminated based on all of the texture attributes, as 20-FRS showed higher intensities for tenderness and juiciness and lower intensities for elasticity, cohesiveness, chewiness, and the attribute screechy as compared with 20-CTL (P < 0.001). The effect of fresh forage feeding on texture attributes of mozzarella can be explained on the basis of the lower SFA content and a fat content that tended to be higher. Both chemical characteristics can contribute to make the cheese softer and stickier (e.g.,
      • Coppa M.
      • Ferlay A.
      • Monsallier F.
      • Verdier-Metz I.
      • Pradel P.
      • Didienne R.
      • Farruggia A.
      • Montel M.C.
      • Martin B.
      Milk fatty acid composition and cheese texture and appearance from cows fed hay or different grazing systems on upland pastures.
      ;
      • Frétin M.
      • Ferlay A.
      • Verdier-Metz I.
      • Fournier F.
      • Montel M.-C.
      • Farruggia A.
      • Delbès C.
      • Martin B.
      The effects of low-input grazing systems and milk pasteurisation on the chemical composition, microbial communities, and sensory properties of uncooked pressed cheeses.
      ).
      Unsurprisingly, the color of mozzarella was unaffected by the diet in both trials, whereas 20-FRS showed a higher uniformity of the external surface and thus was assessed as smoother in terms of appearance in comparison with the corresponding control group (P < 0.01). Numerous authors observed changes in cheese sensory color as a consequence of the ingestion of fresh forage and the related β-carotene content (e.g.,
      • Esposito G.
      • Masucci F.
      • Napolitano F.
      • Braghieri A.
      • Romano R.
      • Manzo N.
      • Di Francia A.
      Fatty acid and sensory profiles of Caciocavallo cheese as affected by management system.
      ). However, these changes could not be observed in buffalo mozzarella cheese, as buffalo milk does not contain detectable amounts of β-carotene (
      • Cerquaglia O.
      • Sottocorno M.
      • Pellegrino L.
      • Ingi M.
      Detection of cow's milk, fat or whey in ewe and buffalo ricotta by HPLC determination of [Beta]-carotene.
      ), due to a more efficient liver enzymatic conversion system of the β-carotene into retinol (
      • Mora O.
      • Romano J.L.
      • Gonzalez E.
      • Ruiz F.J.
      • Shimada A.
      Low cleavage activity of 15,15’ dioxygenase to convert beta-carotene to retinal in cattle compared with goats, is associated with the yellow pigmentation of adipose tissue.
      ).
      Although both products were rated above the neutral point (i.e., 5 = neither pleasant nor unpleasant), the panel composed of untrained and uninformed consumers did not show any preference (P > 0.05) in terms of appearance, taste/flavor, texture, and overall liking (Table 7). These results indicate that consumers were unable to perceive the subtle albeit significant sensory differences detected by the trained panel.
      Table 7Hedonic test (mean ± SE) of Mozzarella cheese obtained from buffalo either fed fresh sorghum (20-FRS) or not (20-CTL)
      Liking
      Mean liking based on a 9-point hedonic scale from “extremely unpleasant” (1) to “extremely pleasant” (9).
      20-CTL20-FRSP-value
      Overall appearance6.51 ± 0.176.85 ± 0.170.158
      Taste/flavor5.69 ± 0.175.87 ± 0.170.460
      Texture6.03 ± 0.186.02 ± 0 180.960
      Overall liking6.56 ± 0.156.58 ± 0.150.923
      1 Mean liking based on a 9-point hedonic scale from “extremely unpleasant” (1) to “extremely pleasant” (9).

      CONCLUSIONS

      This study demonstrated that the inclusion of fresh sorghum in a buffalo TMR at least 26.5% on a DM basis is able to modify the fatty acid composition of buffalo mozzarella cheese. The sensory properties of mozzarella were also modified, whereas no effect on consumer blind acceptance were found. Lower amount of fresh forages (i.e., 13.4% on DM basis) did not have any effects on fat composition or sensory properties of mozzarella. We conclude that, although there are other more effective feeding strategies to modify mozzarella fatty acid profile, fresh-cut forage feeding can represent a low-cost technique to increase the PUFA and CLA content of mozzarella. Further studies are needed to verify whether the information concerning fresh forage feeding may increase mozzarella actual liking, thus providing a tool for product differentiation.

      ACKNOWLEDGMENTS

      The study was supported by Campania Region, PSR 2007-2013 Misura 124, project CEREAMICO (Cereali Micorrizati). Thanks are due to the staff of the farm “Eredi Iemma” (Eboli, Italy) for help with the care of the animals. The technical assistance of A. Guariglia (Department of Agricultural Science, University Federico II, Naples, Italy) is also recognized.

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