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Cheese quality from cows given a tannin extract in 2 different grazing seasons

Open ArchivePublished:June 11, 2021DOI:https://doi.org/10.3168/jds.2021-20292

      ABSTRACT

      The aim of the present study was to compare the effect of dietary tannins on cow cheese quality in 2 different grazing seasons in the Mediterranean. Two experiments were performed on 14 dairy cows reared in an extensive system. The first experiment took place in the wet season (WS), and the second experiment took place in the dry season (DS). In the WS and DS experiments, cows freely grazed green pasture or dry stubbles, respectively, and the diet was supplemented with pelleted concentrate and hay. In both experiments, the cows were divided into 2 balanced groups: a control group and a group (TAN) receiving 150 g of tannin extract/head per day. After 23 d of dietary treatment, individual milk was collected, processed into individual cheeses, and aged 25 d. Milk was analyzed for chemical composition, color parameters, and cheesemaking aptitude (laboratory cheese yield and milk coagulation properties). Cheese was analyzed for chemical composition, proteolysis, color parameters, rheological parameters, fatty acid profile, and odor-active volatile compounds. Data from the WS and DS experiments were statistically analyzed separately with an analysis of covariance model. In the WS experiment, dietary tannin supplementation had no effect on milk and cheese parameters except for a reduced concentration of 2-heptanone in cheese. In the DS experiment, TAN milk showed lower urea N, and TAN cheese had lower C18:1 trans-10 concentration and n-6:n-3 polyunsaturated fatty acid ratio compared with the control group. These differences are likely due to the effect of tannins on rumen N metabolism and fatty acid biohydrogenation. Dietary tannins may differently affect the quality of cheese from Mediterranean grazing cows according to the grazing season. Indeed, tannin bioactivity on rumen metabolism seems to be enhanced during the dry season, when diet is low in protein and rich in acid detergent fiber and lignin. The supplementation dose used in this study (1% of estimated dry matter intake) had no detrimental effects on cheese yield or cheesemaking parameters. Also, it is unlikely that sensorial characteristics would be affected by this kind of dietary tannin supplementation.

      Key words

      INTRODUCTION

      Tannins are a class of polyphenols found in forages, especially in plant species characterizing marginal areas or dry habitats, and in agricultural by-products (
      • Vasta V.
      • Daghio M.
      • Cappucci A.
      • Buccioni A.
      • Serra A.
      • Viti C.
      • Mele M.
      Invited review: Plant polyphenols and rumen microbiota responsible for fatty acid biohydrogenation, fiber digestion, and methane emission: Experimental evidence and methodological approaches.
      ). Thanks to their antimicrobial and protein-binding activities, tannins are known to modify ruminal biohydrogenation (BH) and N metabolism (
      • Patra A.K.
      • Saxena J.
      Exploitation of dietary tannins to improve rumen metabolism and ruminant nutrition.
      ), with positive consequences on milk and cheese quality. Through the impairing of ruminal BH, dietary tannins are often reported to reduce the SFA content and increase the concentration of PUFA, C18:1 trans-11, and C18:2 cis-9,trans-11 in milk (
      • Frutos P.
      • Hervás G.
      • Natalello A.
      • Luciano G.
      • Fondevila M.
      • Priolo A.
      • Toral P.G.
      Ability of tannins to modulate ruminal lipid metabolism and milk and meat fatty acid profiles.
      ). Binding with proteins, tannins can improve the ratio of ruminal protein escape (
      • Waghorn G.
      Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production—Progress and challenges.
      ), increasing both the NAN outflow from the rumen and the EAA concentration of plasma (
      • Min B.R.
      • Attwood G.T.
      • Reilly K.
      • Sun W.
      • Peters J.S.
      • Barry T.N.
      • McNabb W.C.
      Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep.
      ). This could affect cheesemaking properties and cheese quality, as the concentration and type of protein in milk markedly affect cheese yield and its syneretic and rheological characteristics (
      • Guinee T.P.
      Role of protein in cheese and cheese products.
      ). For instance, dietary chestnut tannin extract was found to increase the casein proportion in ewe milk (
      • Buccioni A.
      • Serra A.
      • Minieri S.
      • Mannelli F.
      • Cappucci A.
      • Benvenuti D.
      • Rapaccini S.
      • Conte G.
      • Mele M.
      Milk production, composition, and milk fatty acid profile from grazing sheep fed diets supplemented with chestnut tannin extract and extruded linseed.
      ) and to delay clotting and firming time (
      • Buccioni A.
      • Pauselli M.
      • Minieri S.
      • Roscini V.
      • Mannelli F.
      • Rapaccini S.
      • Lupi P.
      • Conte G.
      • Serra A.
      • Cappucci A.
      • Brufani L.
      • Ciucci F.
      • Mele M.
      Chestnut or quebracho tannins in the diet of grazing ewes supplemented with soybean oil: Effects on animal performances, blood parameters and fatty acid composition of plasma and milk lipids.
      ).
      However, the information available in the literature does not clarify whether and how the effects of dietary tannins on cheese quality might vary according to grazing season in extensive farming systems. Indeed, extensive farming systems are characterized by periods with different forage availability during the year because they strictly depend on climatic conditions that allow grazing or not (
      • Ramírez-Rivera E.J.
      • Rodríguez-Miranda J.
      • Huerta-Mora I.R.
      • Cárdenas-Cágal A.
      • Juárez-Barrientos J.M.
      Tropical milk production systems and milk quality: A review.
      ). This imbalance in diet during the year has well-known implications for animal performance and product quality. In Mediterranean traditional husbandry, dairy cows have higher milk yield, protein content, and fat content during the green season compared with the dry season (
      • Licitra G.
      • Blake R.W.
      • Oltenacu P.A.
      • Barresi S.
      • Scuderi S.
      • Van Soest P.J.
      Assessment of the dairy production needs of cattle owners in southeastern Sicily.
      ). In addition, grazing green pasture is reported to increase the content of vitamins and aromatic compounds (
      • Prache S.
      • Martin B.
      • Coppa M.
      Review: Authentication of grass-fed meat and dairy products from cattle and sheep.
      ) and proportions of PUFA and CLA (
      • Coppa M.
      • Chassaing C.
      • Sibra C.
      • Cornu A.
      • Verbič J.
      • Golecký J.
      • Engel E.
      • Ratel J.
      • Boudon A.
      • Ferlay A.
      • Martin B.
      Forage system is the key driver of mountain milk specificity.
      ) of milk.
      In a recent study, a different response to in vitro rumen BH and fermentation was observed when 2 different tannin extracts were supplemented to a green forage or a hay substrate (
      • Menci R.
      • Coppa M.
      • Torrent A.
      • Natalello A.
      • Valenti B.
      • Luciano G.
      • Priolo A.
      • Niderkorn V.
      Effects of two tannin extracts at different doses in interaction with a green or dry forage substrate on in vitro rumen fermentation and biohydrogenation.
      ). For instance, both the ratio of C18:2 cis-9,trans-11 to C18:2 cis-9,cis-12 and valerate concentration were lower when tannin extracts were included in the hay substrate. Therefore, we hypothesized that dietary tannins could exert different effects on cow cheese quality and composition when supplemented to a green herbage-based diet or a dry forage-based diet. Thus, the aim of the present study was to compare, for the first time, the effect of dietary tannins on cow cheese quality in 2 different grazing seasons in the Mediterranean. We chose to fit into on-farm conditions to directly test the practical effects of dietary tannin extract supplementation. Previous to this study, the response of some of the parameters analyzed, such as rheology and aromatic compounds, to dietary tannin supplementation had never been investigated.

      MATERIALS AND METHODS

      Experimental Design, Animals, and Diets

      All procedures were approved by the animal welfare committee (OPBA) of the University of Catania (UNCTCLE-0015295). Two experiments were conducted in a commercial extensive farm located in the municipality of Ragusa, Italy (36°57′ N, 14°40′ E; altitude: 670 m; annual rainfall: 560 mm), an upland area of the Mediterranean island of Sicily, Italy. The 2 experiments were carried out in different seasons: the first one was conducted in the wet season (WS), and the second one was conducted in the dry season (DS). The WS experiment was performed in the period between March and April 2019, with a total rainfall of 48.5 mm and the temperature ranging from 4 to 18°C (average temperature: 10°C). The DS experiment was performed in July 2019, with a total rainfall of 1.25 mm and the temperature ranging from 15° to 37°C (average temperature: 24.5°C).
      In both experiments, 14 lactating dairy cows (Modicana breed) were used. On the 2 d preceding the beginning of both trials, individual milk was sampled and analyzed. Animals were divided into 2 equivalent groups (n = 7)—control (CON) and tannin (TAN)—balanced for average milk yield (WS: 11.9 ± 1.5 kg/d; DS: 13.6 ± 2.6 kg/d), protein (WS: 40.2 ± 1.3 g/kg; DS: 33.5 ± 1.6 g/kg), and fat (WS: 39.6 ± 2.4 g/kg; DS: 37.2 ± 3.3 g/kg) contents recorded in these 2 d as well as DIM (WS: 190 ± 38 d; DS: 137 ± 43 d), parity (WS: 4 ± 1; DS: 4.2 ± 1), and BCS (WS: 2.8 ± 0.2; DS: 3.6 ± 0.1).
      In WS, the cows were free to graze on 20 ha of spontaneous pasture and had free access to drinking water. Thirty botanical species were identified through botanical surveys of the pasture using the vertical point-quadrat method (
      • Daget P.
      • Poissonet J.
      Une méthode d'analyse phytologique des prairies: Critères d'application.
      ). The main botanical species were Bromus hordaeceus L., Medicago polymorpha L., Lolium perenne L., and Anthemis arvensis L. (17, 13, 12, and 11% on ground cover, respectively). In DS, the cows were free to graze on 20 ha of dry stubble (20-cm harvest cut height because of rocky soil) of an annual crop, composed of vetch (40%), oat (40%), and barley (20%). During the DS experiment, no fresh herbage was available. In both experiments, supplemental pelleted concentrate was individually offered to cows in 2 equal meals just before milking at a rate of 6.4 and 9.6 kg/head per day in WS and DS, respectively. Pelleted concentrate was composed of corn grain (420 g/kg), soybean meal CP 48% (250 g/kg), wheat middlings (100 g/kg), corn flakes (66 g/kg), carob germ (60 g/kg), carob pods (30 g/kg), beet pulp (30 g/kg), rumen-protected fat (10 g/kg; Magnapac, Or Sell S.p.a.), Na2CO3 (10 g/kg), Ca2CO3 (10 g/kg), NaCl (8 g/kg), vitamin and mineral supplement (4 g/kg), and urea (2 g/kg). In addition, in both experiments cows were individually fed supplemental hay (vetch:oat:barley 40:40:20) during milking in 2 equal meals at a rate of 2 kg/head per day. Pelleted concentrate and hay were always completely consumed by all the cows. The chemical composition of feedstuffs is shown in Table 1.
      Table 1Chemical composition of feeds used in the wet season (WS) and dry season (DS) experiments
      ItemConcentrateHayPasture (only WS)Stubble (only DS)
      DM, g/kg889833186876
      Chemical composition, g/kg of DM
       CP2007922269
       Ether extract36122811
       NDF179708415672
       ADF80460269472
       ADL22623876
       Ash51669467
      Phenolic compounds, g of TAeq
      TAeq = tannic acid equivalents.
      /kg of DM
       Phenols5.25.214.25.4
       Tannins3.91.64.71.7
      Protein fraction,
      A = NPN; B1 = buffer-soluble true protein; B2 = neutral detergent soluble protein; B3 = acid detergent soluble protein; C = acid detergent insoluble protein.
      g/100 g of CP
       A15.137.337.122.3
       B17.910.87.618.2
       B259.612.221.420.3
       B312.929.528.226.0
       C4.510.15.813.1
      Fatty acids, g/100 g of fatty acids
       C16:018.229.214.125.1
       C18:09.26.02.46.2
       C18:1 cis-916.89.43.06.5
       C18:2 cis-9,cis-1236.227.012.121.1
       C18:3 cis-9,cis-12,cis-151.816.052.221.6
      1 TAeq = tannic acid equivalents.
      2 A = NPN; B1 = buffer-soluble true protein; B2 = neutral detergent soluble protein; B3 = acid detergent soluble protein; C = acid detergent insoluble protein.
      In both the WS and DS experiments, the TAN group received 150 g/head per day of a commercial tannin extract (Silvafeed ByProX, Silvateam), a 60:40 mixture of chestnut (Castanea sativa Mill.) and quebracho (Schinopsis lorentzii Engl.) tannins, included in pelleted concentrate. Total phenolic compound concentration in tannin extract was 688 g of tannic acid equivalents/kg of DM, with 90.2% of tannins, according to the method of
      • Makkar H.P.S.
      • Blümmel M.
      • Borowy N.K.
      • Becker K.
      Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods.
      . The estimated intake of tannin extract corresponded to 1% of expected DMI based on the potential intake capacity of experimental cows, according to
      • INRA (Institut National de la Recherche Agronomique)
      Alimentation des Ruminants.
      . The feeding trials lasted 23 d.

      Feedstuff Sampling and Analyses

      During the WS and DS experiments, samples of concentrates, hay, pasture, and dry stubble were collected weekly, vacuum packed, and stored at −20°C. For the analyses, the weekly subsamples were pooled to get a representative sample of each feed.
      Ether extract, CP, and ash were determined according to
      • AOAC International
      Official Method of Analysis.
      methods 920.39, 976.06, and 942.05, respectively. Protein fractions were calculated according to the Cornell Net Carbohydrate and Protein System, as modified by
      • Licitra G.
      • Hernandez T.M.
      • Van Soest P.J.
      Standardization of procedures for nitrogen fractionation of ruminant feeds.
      . The analyses of NDF, ADF, and ADL were performed following the method of
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      . Total phenolic compounds and total tannins were analyzed according to the procedure of
      • Makkar H.P.S.
      • Blümmel M.
      • Borowy N.K.
      • Becker K.
      Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods.
      with some modification, as reported by
      • Luciano G.
      • Natalello A.
      • Mattioli S.
      • Pauselli M.
      • Sebastiani B.
      • Niderkorn V.
      • Copani G.
      • Benhissi H.
      • Amanpour A.
      • Valenti B.
      Feeding lambs with silage mixtures of grass, sainfoin and red clover improves meat oxidative stability under high oxidative challenge.
      . Fatty acid (FA) profile of feeds was determined through a 1-step extraction-transesterification with chloroform and sulfuric acid (2% in methanol, vol/vol) as methylation reagent (
      • Valenti B.
      • Luciano G.
      • Pauselli M.
      • Mattioli S.
      • Biondi L.
      • Priolo A.
      • Natalello A.
      • Morbidini L.
      • Lanza M.
      Dried tomato pomace supplementation to reduce lamb concentrate intake: Effects on growth performance and meat quality.
      ). Fatty acid methyl ester separation and quantification was performed using a Thermo Finnigan Trace gas chromatograph equipped with a flame ionization detector (ThermoQuest) and 100-m high-polar fused silica capillary column (SP-2560 fused silica, Supelco; 0.25-mm i.d., 0.25-μm film thickness). Helium was used as carrier gas at a constant flow of 1 mL/min. Oven, injector, and detector were set as described by
      • Natalello A.
      • Luciano G.
      • Morbidini L.
      • Valenti B.
      • Pauselli M.
      • Frutos P.
      • Biondi L.
      • Rufino-Moya P.J.
      • Lanza M.
      • Priolo A.
      Effect of feeding pomegranate byproduct on fatty acid composition of ruminal digesta, liver, and muscle in lambs.
      . Identification of individual FAME was based on comparison with the retention time of commercially available standard FAME mixtures (Nu-Chek Prep Inc., Larodan Fine Chemicals). Individual FA were expressed as grams per 100 grams of total FA.

      Cheesemaking

      In both the WS and DS experiments, 2 cheesemaking sessions were performed, one the day before the beginning of the trial and the other after 23 d of dietary treatment. For each cheesemaking session, 1 pressed and cooked curd cheese (Canestrato type) was made with the individual milk of each experimental cow, for a total of 14 cheeses in each session and 28 cheeses in each experiment. At cheesemaking, individual raw whole milk from the morning milking was pooled with the corresponding refrigerated raw whole milk from the previous evening milking based on individual milk yield. Then, 7 kg of individual raw whole milk was heated in a water bath to reach the optimal temperature for coagulation (38–39°C). A commercial liquid veal rennet [105 international milk clotting units (IMCU)/mL, 80% chymosin and 20% pepsin; Biotec Fermenti s.r.l.] was then added to get a final concentration of 37 IMCU/L, following manufacturer recommendation. One hour later, the curd was cut to a size of 5 to 10 mm, and 1.2 L of water at 75°C was added for curd cooking. Curd was precipitated for 10 min, transferred to a basin, and pressed to remove excess whey. After 20 min of forming, with continuous turning of the curd, a second cooking was carried out by diving forms into 5 L of hot water (75–80°C) for 1 h. Cheeses were then immersed in saturated brine (23 °Bé; 300 g/L NaCl) at 10°C for 2 h. After brining, cheeses were aged in a ripening cellar for 25 d at 10°C and 80% relative humidity. Clotting time and pH were monitored during each cheesemaking step. Weight of cheese was recorded before and after brining and weekly during aging.

      Milk Sampling and Analyses

      Aliquots of the individual milk used for cheesemaking were stored at −20°C pending Ca determination or immediately processed for determination of proximate composition, SCC, color parameters, laboratory cheese yield, and milk coagulation properties. Fat, lactose, and protein contents in milk and MUN were analyzed with a Milkoscan FT 1 (Foss) according to ISO 9622 (
      • ISO. (International Organization for Standardization)
      Milk and liquid milk products—Guidelines for the application of mid-infrared spectrometry. Method no. 9622.
      ). On the same aliquot, SCC was determined using a BacSomatic (Foss) according to ISO 13366-2 (
      • ISO. (International Organization for Standardization)
      Milk—Enumeration of somatic cells—Part 2: Guidance on the operation of fluoro-opto-electronic counters. Method no. 13366-2.
      ). Calcium content in milk was quantified by back titration with EDTA of ashes (
      • Kindstedt P.S.
      • Kosikowski F.V.
      Improved complexometric determination of calcium in cheese.
      ).
      Milk color parameters in the CIE L*a*b space were measured using a Minolta CM-2022 portable spectrophotometer (d/8° geometry; Minolta Co. Ltd.) using illuminant A and 10° standard observer. The reading was performed on a 3-mL milk sample in a 10-mm plastic cuvette. Measured parameters were lightness (L*), redness (a*), yellowness (b*), chroma (C*), hue angle (H*), and the reflectance spectra between 400 and 700 nm. The reflectance spectrum at wavelengths between 450 and 530 nm was used to calculate the integral value I450–530 (
      • Priolo A.
      • Prache S.
      • Micol D.
      • Agabriel J.
      Reflectance spectrum of adipose tissue to trace grass feeding in sheep: Influence of measurement site and shrinkage time after slaughter.
      ).
      Laboratory cheese yield was assessed according to the method of
      • Hurtaud C.
      • Rulquin H.
      • Delaite M.
      • Vérité R.
      Prediction of cheese yielding efficiency of individual milk of dairy cows. Correlation with coagulation parameters and laboratory curd yield.
      . Briefly, 65 µL of the same rennet used for cheesemaking was added to 50 mL of preheated milk in a centrifuge tube and incubated for 1 h at 35°C. After a 5-min pause for syneresis, the coagulum was cut longitudinally using a spatula and centrifuged for 10 min at 2,700 × g at 35°C. The weight of curd after removal of whey was recorded, and laboratory cheese yield was expressed as grams per 100 grams of milk. The individual whey obtained was dried at 100°C, and the laboratory DM cheese yield (LDMCY) was calculated using the following formula (
      • Hurtaud C.
      • Rulquin H.
      • Delaite M.
      • Vérité R.
      Prediction of cheese yielding efficiency of individual milk of dairy cows. Correlation with coagulation parameters and laboratory curd yield.
      ):
      LDMCY = 1 − (dry weight of whey/dry weight of curd and whey).


      The milk coagulation properties of milk at 35°C were analyzed using a milk coagulation meter (Maspres) with a recording interval of 15 s, following the method of
      • Zannoni M.
      • Annibaldi S.
      Standardization of the Renneting Ability of Milk by Formagraph. Pt. 1.
      . Briefly, the same rennet used for cheesemaking was prediluted with water, and 200 µL of the solution was added to 10 mL of milk to obtain a final concentration of 0.046 IMCU/mL. Determined parameters were clotting time (time needed for the beginning of coagulation), firming time (time needed to reach 20 mm of amplitude on the chart), and curd firmness (amplitude of the chart, in mm) after 30 min and after 2 times clotting time.

      Cheese Sampling and Analyses

      After aging, individual cheeses were sampled for analyses and aliquots were vacuum stored at −20°C. Color parameters of the fresh-cut surface of each cheese were measured before storing, and the average value of 3 nonoverlapping zones was recorded. The color parameters and device used were the same as those described for milk analyses.
      Total N and DM were measured according to ISO 5534 (
      • ISO (International Organization for Standardization)
      Cheese and processed cheese—Determination of the total solid content. Method no. 5534.
      ) and ISO 8968-1 (
      • ISO (International Organization for Standardization)
      Milk and milk products—Determination of nitrogen content—Part 1: Kjeldahl principle and crude protein calculation. Method no. 8968-1.
      ), respectively. Water-soluble N and phosphotungstic acid-soluble N were measured using the method proposed by
      • Ardö Y.
      Evaluating proteolysis by analysing the N content of cheese fractions.
      . Calcium content in cheese was quantified by back titration with EDTA of ashes (
      • Kindstedt P.S.
      • Kosikowski F.V.
      Improved complexometric determination of calcium in cheese.
      ). Rheological properties of cheese were assessed by uniaxial compression at constant displacement rate (1 mm/s), as reported by
      • 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.
      .
      Extraction and quantification of fat followed the Röse-Gottlieb method (
      • AOAC
      Official Method of Analysis.
      ), as modified by
      • Secchiari P.
      • Antongiovanni M.
      • Mele M.
      • Serra A.
      • Buccioni A.
      • Ferruzzi G.
      • Paoletti F.
      • Petacchi F.
      Effect of kind of dietary fat on the quality of milk fat from Italian Friesian cows.
      . Fatty acid profile of cheese was then determined through transesterification using a combined basic and acid methylation, as proposed by
      • Cruz-Hernandez C.
      • Deng Z.
      • Zhou J.
      • Hill A.R.
      • Yurawecz M.P.
      • Delmonte P.
      • Mossoba M.M.
      • Dugan M.E.R.
      • Kramer J.K.G.
      Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography.
      . Briefly, the dry lipid extract was dissolved in hexane to get a final concentration of 30 mg/mL. Then, 0.5 mL of lipid extract was incubated at 50°C for 15 min with 1.5 mL of sodium methoxide in methanol (0.5 M). After cooling to room temperature, 1 mL of 5% methanolic HCl was added and the mixture was incubated at 50°C for 30 min. Then, 1 mL of 6% aqueous K2CO3 was added, and a triple centrifugation with 3 mL of hexane at 1,500 × g for 10 min at 4°C was performed. The supernatants collected after each centrifugation were pooled and evaporated under N2 flow at 37°C and then dissolved in 1 mL of GC-grade hexane. The GC setting for FAME identification was the same as described for feedstuff analysis. Moreover, the separation of C18:1 isomers was achieved by isothermal analysis at 165°C. Individual FA were expressed as grams per 100 grams of total FA.
      Odor-active volatile compounds (OAC) were extracted using static solid-phase microextraction as reported by
      • 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.
      , with some modifications. A divinylbenzene/carboxen/polydimethylsiloxane coated fiber (50/30 µm; Supelco) was used to adsorb OAC from the headspace of samples. Samples were prepared by conditioning 10 g of cheese at 40°C for 40 min. An additional 40 min was required for the fiber to establish volatile compound equilibrium between the sample headspace and the fiber solid phase. The fiber was conditioned for 1 h at 225°C before the initial use and for 5 min between each analysis. For the GC-MS analysis and the identification of OAC, a 7890A Series GC system (Agilent Technologies) coupled with an Agilent 5975C Mass Selective Detector (triple axis) was used. An HP-5 capillary column (30 m × 0.25-mm i.d. × 0.25-μm film thickness; Agilent Technologies) was used to separate the volatile components. The chromatographic conditions were as follows: splitless injector at 220°C; oven program conditions: 35°C for 3 min, 6°C/min to 200°C, and 30°C/min to 240°C for 3 min. Carrier gas (helium) pressure and flow were set at 93.77 MPa and 1.0 mL/min, respectively. The mass selective detector operated in scan mode (5.15 scans/s) with 70 eV ionization energy. Peaks were identified by comparison of mass spectra with the bibliographic data of the Wiley 175 library (Wiley and Sons Inc.) and with the linear retention indices of authentic standards (Sigma-Aldrich), calculated by running a paraffin series (from C5 to C20) under the same working conditions. The OAC data were expressed as arbitrary units of chromatograph area.
      For the GC olfactometry analysis, an HP 6890 Series GC system (Agilent Technologies) GC coupled with an olfactometer was used. Column, injection, and oven setting were the same as reported for GC-MS. A trained human nose (sniffer) was used as a final detector simultaneously with the mass detector (
      • Rapisarda T.
      • Pasta C.
      • Carpino S.
      • Caccamo M.
      • Ottaviano M.
      • Licitra G.
      Volatile profile differences between spontaneous and cultivated Hyblean pasture.
      ). The eluted compounds were mixed with humidified air, and the sniffer was continuously exposed to this source for 30 min. During the olfactometric analysis, the sniffer described the perceptions and duration of odors. The OAC recognition was performed using the single sniff method (
      • Marin A.B.
      • Acree T.E.
      • Barnard J.
      Variation in odor detection thresholds determined by charm analysis.
      ). The sniffer was trained with reference chemicals consisting of a group of 8 compounds used to evaluate olfactory acuity. The sniffer had no specific anosmia for these standards.

      Calculations and Statistics

      The index of atherogenicity (IA) and the index of thrombogenicity (IT) were calculated according to
      • Ulbricht T.L.V.
      • Southgate D.A.T.
      Coronary heart disease: Seven dietary factors.
      :
      IA=C12:0+C14:0+C16:0ΣMUFA+ΣPUFA;


      IT=C14:0+C16:0+C18:0ΣMUFA×0.5+Σn-6PUFA×0.5.+Σn-3PUFA+(Σn-3PUFAΣn-6PUFA)


      The hypocholesterolemic to hypercholesterolemic ratio (h:H) was calculated according to
      • Mierliţă D.
      Effects of diets containing hemp seeds or hemp cake on fatty acid composition and oxidative stability of sheep milk.
      :
      h:H=C18:1cis-9+ΣPUFAC12:0+C14:0+C16:0.


      Before statistical analysis, SCC data were transformed to log10 per milliliter, and OAC data were transformed to ln(x + 1) to obtain normalized distribution.
      All data from the WS and DS experiments were analyzed separately using an analysis of covariance (ANCOVA) model of SPSS Statistics 21 (IBM), figuring the fixed effect of dietary treatment (CON, TAN). Individual milk and cheese samples were used as statistical unit. Statistical elaboration was adjusted for a covariate composed by (1) the average data of the 2 d before the beginning of the feeding trials for milk or (2) the data of the day preceding the beginning of the feeding trials for cheese. Differences between means were considered significant at P ≤ 0.05 and a trend toward significance at P ≤ 0.10.

      RESULTS

      WS Experiment

      Table 2 shows the results for milk composition, color parameters, and cheesemaking aptitude, and Table 3 presents data from measurements during cheesemaking, cheese composition, and cheese physical characteristics. Dietary treatment did not affect (P > 0.05) any of these parameters in the WS experiment.
      Table 2Effect of dietary tannin extract supplementation on cow milk yield, chemical composition, color properties, and cheesemaking aptitude in the wet season (WS) and dry season (DS) experiments
      ItemWSDS
      Treatment
      CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      SEMP-valueTreatmentSEMP-value
      CONTANCONTAN
      Milk yield, kg/d10.311.10.550.47912.312.30.200.969
      Protein, g/kg39.239.90.830.70333.232.20.280.137
      Fat, g/kg39.339.51.260.94234.838.41.700.318
      Lactose, g/kg45.846.10.620.81646.646.10.320.422
      Calcium, g/kg1.431.710.1300.1361.431.000.1700.100
      SCC, log10/mL2.852.970.0500.2842.802.970.0480.119
      MUN, mg/dL33.234.20.740.53329.625.41.090.031
      Color parameter
      L* = lightness; a* = redness; b* = yellowness; C* = chroma; H* = hue angle; I450–530 = integral value of the absorbance spectrum between 450 and 530 nm.
       L*70.971.50.330.37768.869.70.330.198
       a*−1.44−1.390.0900.752−2.00−1.800.1140.405
       b*3.803.810.3290.994−0.1490.6590.2110.091
       C*4.104.150.2780.9282.742.830.1780.827
       H*1131132.90.9841651595.00.554
       I450–530−289−25820.40.347−48.9−92.719.210.144
      Cheesemaking aptitude
      LCY = laboratory cheese yield; LDMCY = laboratory DM cheese yield; R = clotting time; K20 = firming time; A30 = curd firmness after 30 min; A2R = curd firmness after 2 times R.
       LCY, g/100 g26.026.00.010.97323.725.00.010.347
       LDMCY, g/100 g8.608.700.0020.7507.207.800.0020.238
       R, min:s22:4524:141:180.59020:4921:432:040.842
       K20, min:s6:046:010:540.9844:534:040:130.133
       A30, mm39.227.15.000.28332.224.64.530.467
       A2R, mm47.045.72.730.82537.337.31.070.966
      1 CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      2 L* = lightness; a* = redness; b* = yellowness; C* = chroma; H* = hue angle; I450–530 = integral value of the absorbance spectrum between 450 and 530 nm.
      3 LCY = laboratory cheese yield; LDMCY = laboratory DM cheese yield; R = clotting time; K20 = firming time; A30 = curd firmness after 30 min; A2R = curd firmness after 2 times R.
      Table 3Effect of dietary tannin extract supplementation on cow cheese composition, color properties, rheological properties, and cheesemaking measurements in the wet season (WS) and dry season (DS) experiments
      ItemWSDS
      Treatment
      CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      SEMP-valueTreatmentSEMP-value
      CONTANCONTAN
      Cheesemaking measurement
       Cheese yield, g/kg112.5112.83.470.97883.983.52.360.931
       Milk pH6.686.680.0150.8856.546.560.0090.216
       pH after first curd cooking6.656.600.0220.2316.506.510.0100.670
       pH after second curd cooking6.546.550.0250.8526.396.370.0270.657
       Weight after first curd cooking, g1,5051,51370.70.9601,2181,10029.40.075
       Weight after second curd cooking, g1,0311,08346.50.59187478629.40.166
       Weight before brining, g94995232.70.96874168019.40.164
       Weight after brining, g95095833.10.90774368319.40.167
      Composition
      TN = total N; WSN = water-soluble N; PTASN = phosphotungstic acid-soluble N.
       DM, g/100 g55.456.20.470.35863.362.40.950.643
       Fat, g/100 g17.818.40.800.68819.720.50.920.691
       Fat, g/100 g of DM31.832.71.150.71130.732.91.360.438
       TN, g/100 g of DM6.896.630.0850.1596.756.410.1510.289
       WSN, g/100 g of DM0.8020.7450.0550.6170.6010.7350.0510.223
       PTASN, g/100 g of DM0.5460.5150.0510.7660.4710.5420.0480.492
       Calcium, g/100 g0.8450.9130.0500.3691.081.010.0270.135
      Color parameter
      L* = lightness; a* = redness; b* = yellowness; C* = chroma; H* = hue angle; I450–530 = integral value of the absorbance spectrum between 450 and 530 nm.
       L*83.283.80.890.72277.079.90.940.057
       a*3.763.700.1110.7831.701.930.1060.154
       b*19.920.40.600.6799.5710.210.4200.306
       C*20.220.80.610.6989.7210.400.4250.288
       H*79.479.60.110.40679.679.70.280.757
       I450–530−1,152−1,21137.70.493−438−50932.40.156
      Rheology, N/cm2
       Strength to 20% deformation2.182.600.2360.2346.535.581.0030.523
       Strength to 40% deformation6.026.890.6140.33915.612.81.880.324
       Strength to 60% deformation8.299.470.6700.24320.816.62.290.231
       Young modulus (undeformability)25.027.72.000.35863.647.911.900.372
      1 CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      2 TN = total N; WSN = water-soluble N; PTASN = phosphotungstic acid-soluble N.
      3 L* = lightness; a* = redness; b* = yellowness; C* = chroma; H* = hue angle; I450–530 = integral value of the absorbance spectrum between 450 and 530 nm.
      Fatty acids proportion in cheese is reported in Table 4. In the WS experiment, cheese from the TAN group did not differ (P > 0.05) from cheese from the CON group, except that it had a higher (P < 0.001) proportion of C20:3 n-6. Supplementation with tannin extract did not affect (P > 0.05) BH and healthfulness indices after 23 d of dietary treatment. A more detailed FA profile is given in Supplemental Table S1.
      Table 4Effect of dietary tannin extract supplementation on cow cheese fatty acid (FA) profile (g/100 g of total FA) in the wet season (WS) and dry season (DS) experiments
      Item
      de novo FA = C4:0 + C6:0 + C8:0 + C10:0 + C12:0 + C14:0; OBCFA = odd- and branched-chain fatty acids; BHI = biohydrogenation intermediates; DSI C14 = desaturation index, calculated as C14:1 cis-9/(C14:0 + C14:1 cis-9); IA = index of atherogenicity (Ulbricht and Southgate, 1991); IT = index of thrombogenicity (Ulbricht and Southgate, 1991); h:H = hypocholesterolemic:hypercolesterolemic ratio (Mierliţă, 2018); LA = linoleic acid; RA = rumenic acid.
      WSDS
      Treatment
      CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      SEMP-valueTreatmentSEMP-value
      CONTANCONTAN
      Σ de novo FA19.6518.291.1300.41315.7514.870.8710.484
       C16:026.8628.900.7510.21029.6529.900.4080.799
       C16:1 cis-91.301.260.0610.7151.341.430.0560.509
       C18:010.9511.060.3790.88411.5811.150.4910.780
       C18:1 trans-90.320.310.0240.8010.360.380.0220.656
       C18:1 trans-100.290.310.0170.6940.350.270.0120.010
       C18:1 trans-113.613.670.1560.8532.082.230.0420.133
       C18:1 cis-921.2520.620.7690.68724.1925.490.3740.128
       C18:1 cis-111.681.700.0900.9352.242.220.0990.925
       C18:2 cis-9,cis-12 (LA)1.922.020.0820.5582.482.440.0840.793
       C18:2 cis-9,trans-11 (RA)1.371.330.0990.8700.660.680.0480.872
       C18:3 cis-9,cis-12,cis-151.040.920.0820.4810.280.390.0260.054
       C20:00.120.120.0100.9490.160.170.0140.589
       C20:5 n-30.060.070.0110.4280.050.060.0120.883
       C22:00.110.180.0210.1160.090.080.0090.818
       C22:5 n-30.040.070.0080.1180.060.060.0150.805
       C24:00.040.040.0080.8930.030.030.0080.991
      Σ SFA57.3858.951.1040.49557.1056.390.4980.511
      Σ MUFA31.7330.880.8970.64633.5234.680.3910.187
      Σ PUFA5.185.180.2430.9923.894.120.1550.488
      Σ OBCFA5.595.300.1720.5425.335.200.1380.659
      SFA/PUFA11.311.80.6540.70914.9414.100.6470.549
      Σ n-6 PUFA2.092.250.0850.3692.7272.7200.0900.970
      Σ n-3 PUFA1.131.080.0810.7440.3810.5010.0310.092
      n-6:n-3 PUFA1.862.520.3050.3047.495.640.3700.031
      C18:1 trans-11/trans-1013.012.00.7920.5466.068.390.4610.033
      BHI9.999.730.4120.7587.797.920.1760.753
      RA/LA0.7280.6570.0440.4430.2720.2880.0240.749
      DSI C140.0660.0560.0050.3250.0600.0610.0030.924
      IA2.242.350.1690.6342.061.920.1050.373
      IT2.432.650.1530.3372.712.550.0900.241
      h:H0.6250.5760.0540.5310.6550.6990.0300.347
      1 de novo FA = C4:0 + C6:0 + C8:0 + C10:0 + C12:0 + C14:0; OBCFA = odd- and branched-chain fatty acids; BHI = biohydrogenation intermediates; DSI C14 = desaturation index, calculated as C14:1 cis-9/(C14:0 + C14:1 cis-9); IA = index of atherogenicity (
      • Ulbricht T.L.V.
      • Southgate D.A.T.
      Coronary heart disease: Seven dietary factors.
      ); IT = index of thrombogenicity (
      • Ulbricht T.L.V.
      • Southgate D.A.T.
      Coronary heart disease: Seven dietary factors.
      ); h:H = hypocholesterolemic:hypercolesterolemic ratio (
      • Mierliţă D.
      Effects of diets containing hemp seeds or hemp cake on fatty acid composition and oxidative stability of sheep milk.
      ); LA = linoleic acid; RA = rumenic acid.
      2 CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      We detected up to 61 different OAC in cheeses from the WS experiment, belonging to the following chemical classes: acids (8), alcohols (13), aldehydes (4), aromatic hydrocarbons (7), esters (9), ketones (8), sulfurs (5), and terpenes (7). However, not all of these OAC were detected in every cheese sample. Therefore, Table 5 shows only the OAC for which statistical analysis could be performed. 2-Nonanone (ketones), ethyl hexanoate (ester), and hexanoic and octanoic acid were the main OAC in WS cheeses. 2-Heptanone was the most abundant compound in CON cheese, but dietary tannin extract decreased (P = 0.018) its concentration.
      Table 5Effect of dietary tannin extract supplementation on proportion of cow cheese odor-active volatile compounds
      All OAC reported in this table were identified using both an Agilent 5975C Mass Selective Detector and the bibliographic data of the Wiley 175 library (Wiley and Sons Inc.).
      (OAC) in wet season (WS) and dry season (DS) experiments (expressed as arbitrary units of chromatograph area)
      ItemOdor perceptionLRI
      LRI = linear retention index.
      WSDS
      Treatment
      CON = control group; TAN = group receiving 150 g of tannin extract/head per day.
      SEMP-valueTreatmentSEMP-value
      CONTANCONTAN
      Acids, total17.416.90.270.34516.316.30.800.895
       Butanoic acidCheese82015.413.90.770.34212.510.61.120.413
       3-Methylbutanoic acidRancid, cheese87714.514.50.920.934
       Hexanoic acidSweat1,01917.016.50.730.47014.615.61.290.126
       Octanoic acidCheese1,27916.015.50.650.12713.713.80.940.984
       Decanoic acidRancid1,37314.311.51.040.19811.510.91.280.803
      Alcohols, total13.113.90.870.66513.413.60.890.875
       3-Methylacetatebutan-1-olBanana87610.811.91.060.630
       2-Ethylhexan-1-olRose, green1,03211.011.31.110.88911.312.80.850.401
      Aldehydes, total10.510.51.020.9818.910.30.990.498
       NonanalFresh, green1,10410.510.51.020.9978.810.30.990.464
      Aromatic hydrocarbons, total15.916.30.230.52614.314.30.630.924
       BenzeneacetaldehydeHoney1,04910.610.50.920.9718.510.10.900.392
       Phenylethyl alcoholRose, honey, orange1,11813.814.10.210.46513.111.70.790.388
       ToluenePaint77315.515.60.380.969
      Esters, total16.216.30.340.86014.615.01.160.856
       Ethyl butanoateApple80412.713.41.060.78115.015.81.080.228
       Ethyl hexanoateOrange1,00015.916.40.710.29514.314.71.100.884
       Ethyl octanoateWine1,19814.914.80.600.96911.513.61.060.348
       Ethyl decanoateGrape1,39812.410.30.770.20010.612.10.840.421
       Ketones, total18.216.70.350.07818.018.00.430.990
       2-HeptanoneSoap, fruit89518.214.00.660.01818.217.80.470.534
       2-OctanoneSolvent99912.612.40.900.92414.413.80.950.246
       2-NonanoneHot milk1,09317.015.90.430.26717.117.20.410.836
       2-UndecanoneOrange1,29613.113.00.880.944
       Terpenes, total9.911.40.960.441
       α-PineneFresh9399.09.70.950.726
      1 All OAC reported in this table were identified using both an Agilent 5975C Mass Selective Detector and the bibliographic data of the Wiley 175 library (Wiley and Sons Inc.).
      2 LRI = linear retention index.
      3 CON = control group; TAN = group receiving 150 g of tannin extract/head per day.

      DS Experiment

      Concerning milk characteristics (Table 2), only MUN differed (P = 0.031) between treatments, being lower in the TAN group than in the CON group. Tannin supplementation did not exert any effect (P > 0.05) on cheesemaking parameters, cheese composition, and physical characteristics (Table 3) in the DS experiment. The color of cheeses only slightly differed for luminosity, with the TAN group showing a tendency for a higher (P = 0.057) L* value than CON.
      Concerning FA profile (Table 4), dietary tannin extract decreased (P = 0.010) C18:1 trans-10 concentration and tended to increase (P = 0.054) C18:3 cis-9,cis-12,cis-15 concentration. Consequently, we found higher (P = 0.033) C18:1 trans-11 to C18:1 trans-10 ratio and lower (P = 0.031) n-6 PUFA to n-3 PUFA ratio (n-6:n-3) in cheeses from the TAN group. A more detailed FA profile is given in Supplemental Table S1 (http://dx.doi.org/10.17632/bb4xjj3fbt.1).
      We detected up to 41 different OAC in cheeses from the DS experiment, belonging to the following chemical classes: acids (5), alcohols (7), aldehydes (2), aromatic hydrocarbons (5), esters (9), ketones (7), sulfurs (2), and terpenes (4). However, not all of these OAC were detected in every cheese sample. Therefore, Table 5 shows only the OAC for which statistical analysis could be performed. Ketones were the most abundant compounds in all the cheeses, especially 2-heptanone and 2-nonanone, followed by ethyl butanoate (ester) and hexanoic acid. We found no differences (P > 0.05) in the OAC composition between the cheeses of the 2 dietary groups.

      DISCUSSION

      Effect on Parameters Related to N Metabolism

      One of the starting hypotheses of this study was that tannins, thanks to their well-known protein-binding and antimicrobial activity (
      • Patra A.K.
      • Saxena J.
      Exploitation of dietary tannins to improve rumen metabolism and ruminant nutrition.
      ), could affect N metabolism in vivo and consequently modify some of the cheese parameters related to protein content and composition. However, this occurred only in the DS experiment and was limited to MUN, with no consequences on protein content, proteolysis, or clotting and rheology parameters.
      The reduction of MUN in ruminants eating tannins from either extracts or forages is reported in several studies on dairy cows (
      • Broderick G.A.
      • Grabber J.H.
      • Muck R.E.
      • Hymes-Fecht U.C.
      Replacing alfalfa silage with tannin-containing birdsfoot trefoil silage in total mixed rations for lactating dairy cows.
      ;
      • Zhang J.
      • Xu X.
      • Cao Z.
      • Wang Y.
      • Yang H.
      • Azarfar A.
      • Li S.
      Effect of different tannin sources on nutrient intake, digestibility, performance, nitrogen utilization, and blood parameters in dairy cows.
      ;
      • Aguerre M.J.
      • Duval B.
      • Powell J.M.
      • Vadas P.A.
      • Wattiaux M.A.
      Effects of feeding a quebracho–chestnut tannin extract on lactating cow performance and nitrogen utilization efficiency.
      ) and ewes (
      • Buccioni A.
      • Serra A.
      • Minieri S.
      • Mannelli F.
      • Cappucci A.
      • Benvenuti D.
      • Rapaccini S.
      • Conte G.
      • Mele M.
      Milk production, composition, and milk fatty acid profile from grazing sheep fed diets supplemented with chestnut tannin extract and extruded linseed.
      ;
      • Maamouri O.
      • Mahouachi M.
      • Kraiem K.
      • Atti N.
      Milk production, composition and milk fatty acid profile from grazing ewes fed diets supplemented with Acacia cyanophylla leaves as tannins source and whole or extruded linseed.
      ). This phenomenon is often combined with a lower ureic N concentration in urine because it is due to an impaired protein ruminal degradation that decreases the concentration of ammonia in the rumen (
      • Patra A.K.
      • Saxena J.
      Exploitation of dietary tannins to improve rumen metabolism and ruminant nutrition.
      ). As a consequence, ammonia conversion to urea in the liver and subsequent ureic emission from the ruminant are reduced as well as the nitrous oxide emissions from manure, with positive implications for the environment (
      • Naumann H.D.
      • Tedeschi L.O.
      • Zeller W.E.
      • Huntley N.F.
      The role of condensed tannins in ruminant animal production: Advances, limitations and future directions.
      ). This effect is desirable in the WS, when young green herbage is rich in degradable protein, which may cause a surplus of soluble N in the rumen (
      • Kingston-Smith A.H.
      • Theodorou M.K.
      Tansley review no. 118: Post-ingestion metabolism of fresh forage.
      ). In the WS experiment in the present study, the lack of reduction in MUN in the TAN group may be due to the high level of CP intake from green pasture, which hid the effect of tannin extract supplementation at the dosage used here. A higher proportion of tannins in DMI likely could have resulted in a significant effect in the WS experiment, as the effect of tannins on rumen fermentation is often dose dependent (
      • Toral P.G.
      • Monahan F.J.
      • Hervás G.
      • Frutos P.
      • Moloney A.P.
      Modulating ruminal lipid metabolism to improve the fatty acid composition of meat and milk. Challenges and opportunities.
      ). However, a high intake of tannins could have detrimental consequences on animal performance (
      • Aguerre M.J.
      • Capozzolo M.C.
      • Lencioni P.
      • Cabral C.
      • Wattiaux M.A.
      Effect of quebracho-chestnut tannin extracts at 2 dietary crude protein levels on performance, rumen fermentation, and nitrogen partitioning in dairy cows.
      ) and could be economically impractical on a commercial farm.
      On the other hand, the literature lacks studies assessing the effect of dietary tannins on the physical properties of cow cheese or the cheesemaking aptitude of cow milk.
      • Kälber T.
      • Kreuzer M.
      • Leiber F.
      Effect of feeding buckwheat and chicory silages on fatty acid profile and cheese-making properties of milk from dairy cows.
      found that milk of cows eating 6.1 g/d of condensed tannins from buckwheat (Fagopyrum esculentum Moench) forage had a shorter clotting time compared with milk of cows eating 2.2 g/d of condensed tannins from chicory (Cichorium intybus L.) or ryegrass (Lolium multiflorum Lam.) forage. However, the authors did not observe any differences in milk composition that could explain that positive result. In another study, a trained panel noted moderate differences in hardness and adhesiveness of Gruyère cheese from Holstein cows eating 691 g/head per day of condensed tannins from sainfoin (Onobrychis viciifolia Scop.) pellets, but no difference in protein or casein content was observed in the milk used for cheesemaking (
      • Girard M.
      • Dohme-Meier F.
      • Wechsler D.
      • Goy D.
      • Kreuzer M.
      • Bee G.
      Ability of 3 tanniferous forage legumes to modify quality of milk and Gruyère-type cheese.
      ). Additional information can be collected from experiments on small ruminants, although caution is needed when knowledge is extrapolated from different ruminant species, as it is a relevant source of variation. Some articles reported no effect of dietary quebracho or chestnut tannin extracts on ewe milk clotting parameters (
      • Buccioni A.
      • Pauselli M.
      • Viti C.
      • Minieri S.
      • Pallara G.
      • Roscini V.
      • Rapaccini S.
      • Marinucci M.T.
      • Lupi P.
      • Conte G.
      • Mele M.
      Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes.
      ,
      • Buccioni A.
      • Serra A.
      • Minieri S.
      • Mannelli F.
      • Cappucci A.
      • Benvenuti D.
      • Rapaccini S.
      • Conte G.
      • Mele M.
      Milk production, composition, and milk fatty acid profile from grazing sheep fed diets supplemented with chestnut tannin extract and extruded linseed.
      ) or even longer clotting and firming time (
      • Buccioni A.
      • Pauselli M.
      • Minieri S.
      • Roscini V.
      • Mannelli F.
      • Rapaccini S.
      • Lupi P.
      • Conte G.
      • Serra A.
      • Cappucci A.
      • Brufani L.
      • Ciucci F.
      • Mele M.
      Chestnut or quebracho tannins in the diet of grazing ewes supplemented with soybean oil: Effects on animal performances, blood parameters and fatty acid composition of plasma and milk lipids.
      ), probably related to the interference with caseins or rennin of some bioactive monomers derived from tannin biodegradation.
      • Bonanno A.
      • Di Grigoli A.
      • Mazza F.
      • De Pasquale C.
      • Giosuè C.
      • Vitale F.
      • Alabiso M.
      Effects of ewes grazing sulla or ryegrass pasture for different daily durations on forage intake, milk production and fatty acid composition of cheese.
      observed a positive effect on ewe milk and cheese protein content when animals grazed on sulla (Sulla coronaria Medik.) compared with ryegrass. These results were attributed to the presence of condensed tannins in sulla; however, animals grazing ryegrass had a significantly lower CP daily intake (−53%). In summary, a recent meta-analysis by
      • Herremans S.
      • Vanwindekens F.
      • Decruyenaere V.
      • Beckers Y.
      • Froidmont E.
      Effect of dietary tannins on milk yield and composition, nitrogen partitioning and nitrogen use efficiency of lactating dairy cows: A meta-analysis.
      concluded that dietary tannins do not have any effect on N use efficiency in dairy cattle, except for the reduction of urea emissions, which is beneficial for the environment.

      Effect on FA Profile

      The significant reduction of n-6:n-3 in 25-d-old cheeses from cows ingesting tannin extract in the DS experiment is most likely related to the effect of tannins on rumen BH. Indeed, the milk FA synthesized de novo in animal tissues are mainly SFA, or they result from the activity of desaturase and elongase enzymes (
      • Palmquist D.L.
      Milk fat: Origin of fatty acids and influence of nutritional factors thereon.
      ). Milk C18:2 cis-9,cis-12 and C18:3 cis-9,cis-12,cis-15, the most relevant n-6 and n-3 PUFA, respectively, are mainly of dietary origin, and their concentration can be modified by modulating BH (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ). A lower dietary n-6:n-3 could reduce cardiovascular disease risk in human, especially if achieved as a result of an increase in C18:3 cis-9,cis-12,cis-15 concentration (
      • Harris W.S.
      The omega-6/omega-3 ratio and cardiovascular disease risk: Uses and abuses.
      ), as occurred in our study. Likewise,
      • Girard M.
      • Dohme-Meier F.
      • Wechsler D.
      • Goy D.
      • Kreuzer M.
      • Bee G.
      Ability of 3 tanniferous forage legumes to modify quality of milk and Gruyère-type cheese.
      reported an increase of 17% of C18:3 cis-9,cis-12,cis-15 proportion in cheese from cows fed sainfoin pellets (691 g of condensed tannins/head per day) compared with the control group (alfalfa, Medicago sativa L.). Also, dietary tannins were suggested to be responsible for the increase in C18:3 cis-9,cis-12,cis-15 in 60-d-old cheese from ewes fed fresh sulla forage, although tannin content in diets was not investigated (
      • Addis M.
      • Cabiddu A.
      • Pinna G.
      • Decandia M.
      • Piredda G.
      • Pirisi A.
      • Molle G.
      Milk and cheese fatty acid composition in sheep fed Mediterranean forages with reference to conjugated linoleic acid cis-9,trans-11.
      ). Unfortunately, the tendentially higher n-3 PUFA concentration found in TAN in the present study cheese was not enough to improve the healthfulness indices (i.e., IA, IT, and h:H).
      When the effects of dietary tannins are investigated in vivo, a shift in milk C18:3 cis-9,cis-12,cis-15 concentration is generally combined with changes in the concentration of other FA involved in BH, such as C18:2 cis-9,cis-12, C18:0, C18:1 trans-11, and C18:2 cis-9,trans-11 (
      • Cabiddu A.
      • Molle G.
      • Decandia M.
      • Spada S.
      • Fiori M.
      • Piredda G.
      • Addis M.
      Responses to condensed tannins of flowering sulla (Hedysarum coronarium L.) grazed by dairy sheep. Part 2: Effects on milk fatty acid profile.
      ;
      • Buccioni A.
      • Pauselli M.
      • Viti C.
      • Minieri S.
      • Pallara G.
      • Roscini V.
      • Rapaccini S.
      • Marinucci M.T.
      • Lupi P.
      • Conte G.
      • Mele M.
      Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes.
      ,
      • Buccioni A.
      • Serra A.
      • Minieri S.
      • Mannelli F.
      • Cappucci A.
      • Benvenuti D.
      • Rapaccini S.
      • Conte G.
      • Mele M.
      Milk production, composition, and milk fatty acid profile from grazing sheep fed diets supplemented with chestnut tannin extract and extruded linseed.
      ). However,
      • Frutos P.
      • Hervás G.
      • Natalello A.
      • Luciano G.
      • Fondevila M.
      • Priolo A.
      • Toral P.G.
      Ability of tannins to modulate ruminal lipid metabolism and milk and meat fatty acid profiles.
      summarized in a recent review that although dietary tannins are commonly found to increase C18:3 cis-9,cis-12,cis-15 concentration in milk, regardless of the tannin source, the effects on C18:2 cis-9,cis-12 and C18:0 are less consistent. In the DS experiment, dietary tannin extract exerted no effect on these protagonists of BH, but we observed a significant decrease in C18:1 trans-10 concentration. As the microbial conversion of C18:3 cis-9,cis-12,cis-15 to C18:1 trans-10 may occur in the rumen (
      • Bessa R.J.B.
      • Alves S.P.
      • Santos-Silva J.
      Constraints and potentials for the nutritional modulation of the fatty acid composition of ruminant meat.
      ), dietary tannin extract may have affected this particular pathway in the DS experiment. An increase in C18:3 cis-9,cis-12,cis-15 concentration and a reduction of C18:1 trans-10 concentration in milk due to tannin ingestion (26.5 g of condensed tannins/kg of DMI) was also reported by
      • Cabiddu A.
      • Molle G.
      • Decandia M.
      • Spada S.
      • Fiori M.
      • Piredda G.
      • Addis M.
      Responses to condensed tannins of flowering sulla (Hedysarum coronarium L.) grazed by dairy sheep. Part 2: Effects on milk fatty acid profile.
      in ewes eating fresh sulla, but this effect was also combined with changes in other FA concentrations. Other studies on dietary tannin supplementation to dairy cows reported no effect on C18:1 trans-10 concentration in cow milk (
      • Dschaak C.M.
      • Williams C.M.
      • Holt M.S.
      • Eun J.S.
      • Young A.J.
      • Min B.R.
      Effects of supplementing condensed tannin extract on intake, digestion, ruminal fermentation, and milk production of lactating dairy cows.
      ;
      • Henke A.
      • Westreicher-Kristen E.
      • Molkentin J.
      • Dickhoefer U.
      • Knappstein K.
      • Hasler M.
      • Susenbeth A.
      Effect of dietary quebracho tannin extract on milk fatty acid composition in cows.
      ), but it should be emphasized that this FA is often not reported in scientific articles or its concentration is summed with that of C18:1 trans-11, as they easily coelute in GC.
      Conversely, it seems that dietary tannin supplementation did not affect ruminal BH in the WS experiment according to cheese FA profile. In a study comparing the effect of 2 different tannin extracts (quebracho vs. chestnut and quebracho) on FA profile of in vitro rumen fermentation with different forage substrates, hay was found to be more susceptible than green herbage to tannin bioactivity (
      • Menci R.
      • Coppa M.
      • Torrent A.
      • Natalello A.
      • Valenti B.
      • Luciano G.
      • Priolo A.
      • Niderkorn V.
      Effects of two tannin extracts at different doses in interaction with a green or dry forage substrate on in vitro rumen fermentation and biohydrogenation.
      ). Because diet is likely the major factor affecting rumen microbiota composition (
      • Ellison M.J.
      • Conant G.C.
      • Lamberson W.R.
      • Cockrum R.R.
      • Austin K.J.
      • Rule D.C.
      • Cammack K.M.
      Diet and feed efficiency status affect rumen microbial profiles of sheep.
      ), the low protein content or the high structural carbohydrate content of dry forages or both may select a particular microbiota that is more sensitive to the effects of tannin. The results of the present study confirm this phenomenon in vivo with cows, considering that ruminal microorganisms are among the main factors responsible for FA profile of milk and dairy (
      • Palmquist D.L.
      Milk fat: Origin of fatty acids and influence of nutritional factors thereon.
      ). Indeed, the diet in the DS experiment was poorer in CP and richer in ADL compared with the WS experiment, as the chemical composition of the stubble grazed by cows was comparable with that of hay. Further studies on rumen microbiota involving different tannin sources and types are needed to confirm our hypothesis.

      Effect on OAC

      Dietary tannin extract supplementation did not exert any effect on the aroma of DS cheeses, but it did affect ketone concentration in the WS experiment, particularly 2-heptanone concentration. Methyl ketones originate from the β-oxidation of lipolyzed FA by microorganisms (
      • McSweeney P.L.H.
      • Sousa M.J.
      Biochemical pathways for the production of flavour compounds in cheeses during ripening: A review.
      ); therefore, they are known to characterize the aroma of blue and surface-mold ripened cheeses. Their presence is also reported in different kinds of cheese (
      • Curioni P.M.G.
      • Bosset J.O.
      Key odorants in various cheese types as determined by gas chromatography-olfactometry.
      ). Interestingly, herd management affected the presence of ketones in cheese.
      • Valdivielso I.
      • Bustamante M.A.
      • Aldezabal A.
      • Amores G.
      • Virto M.
      • Ruiz de Gordoa J.C.
      • de Renobales M.
      • Barron L.J.R.
      Case study of a commercial sheep flock under extensive mountain grazing: Pasture derived lipid compounds in milk and cheese.
      observed a significant increase of 2-heptanone and 2-nonanone in cheese from ewes grazing mountain pastures, and
      • 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.
      found acetoin and 2-nonanone only in cheese from grazing cows even though none of these compounds were detected in pasture. At the moment, it is not clear how dietary tannins affected ketone concentration or whether they acted against lipolysis or β-oxidation enzymes. Further research should investigate this aspect, as the literature is devoid of studies on the effect of dietary tannins on aromatic compounds in milk and cheese. In summary, the slight differences in OAC concentrations of cow cheese induced by dietary tannin extract at the dose used in this study would likely not affect the consumer sensory experience.

      CONCLUSIONS

      The inclusion of tannin extract in the diet of dairy cows at a rate of 1% of estimated DMI for 23 d slightly affected the composition and OAC of cow cheese. The results of the present study indicated that this rate of tannin supplementation has no detrimental effects on cheese yield or other cheesemaking parameters regardless of the variation in cow diet induced by forage availability according to the season of the Mediterranean climate. The bioactivity of dietary tannins appears to be more efficient during the DS, when the diet is low in CP and rich in ADF and ADL. Further studies are needed to investigate the effects of longer supplementations or different doses and tannin sources.

      ACKNOWLEDGMENTS

      The authors acknowledge the financial support provided by transnational funding bodies that are partners of the H2020 ERA-net project “CORE Organic Cofund” and the cofund from the European Commission, under the project ProYoungStock “Promoting young stock and cow health and welfare by natural feeding systems.” Moreover, the authors acknowledge the University of Catania (Catania, Italy) for funding part of the research conducted (project “QUALIGEN”; Linea 2 – Piano di Incentivi per la Ricerca di Ateneo 2020/2022; principal investigator Giuseppe Luciano). R. Menci was granted fellowship by Programma Operativo Nazionale Ricerca e Innovazione 2014-2020, “Dottorati Innovativi con caratterizzazione Industriale” Borsa di studio DOT1308937-1 – CUP: E67I18001070006, PhD course in Agricultural, Food and Environmental Science of the University of Catania. The authors also acknowledge Silvateam s.p.a. (San Michele Mondovì, Cuneo, Italy) for providing the tannin extracts used in this experiment. The authors declare no conflicts of interest.

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