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Fatty Acid and Cholestrol Content ofManchego Type Cheese Prepared withIncorporated Avocado OilManuel Algarra a , Cristobal Sánchez b , Joaquim C.G. Esteves daSilva c & José Jiménez-Jiménez da Centro de Geología Porto, Faculty of Science, University of Porto,Porto, Portugalb Departament of Analytical Chemistry, Faculty of Science,University of Málaga, Málaga, Spainc Centro de Investigação em Química (CIQ), Faculty of Science,University of Porto, Porto, Portugald Departament of Inorganic Chemistry, Faculty of Science, Universityof Málaga, Málaga, Spain

Available online: 24 Jun 2011

To cite this article: Manuel Algarra, Cristobal Sánchez, Joaquim C.G. Esteves da Silva & JoséJiménez-Jiménez (2012): Fatty Acid and Cholestrol Content of Manchego Type Cheese Prepared withIncorporated Avocado Oil, International Journal of Food Properties, 15:4, 796-808

To link to this article: http://dx.doi.org/10.1080/10942912.2010.503358

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International Journal of Food Properties, 15:796–808, 2012Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942912.2010.503358

FATTY ACID AND CHOLESTROL CONTENT OFMANCHEGO TYPE CHEESE PREPARED WITHINCORPORATED AVOCADO OIL

Manuel Algarra1, Cristobal Sánchez2,Joaquim C.G. Esteves da Silva3, and José Jiménez-Jiménez4

1Centro de Geología Porto, Faculty of Science, University of Porto,Porto, Portugal2Departament of Analytical Chemistry, Faculty of Science, University of Málaga,Málaga, Spain3Centro de Investigação em Química (CIQ), Faculty of Science, University ofPorto, Porto, Portugal4Departament of Inorganic Chemistry, Faculty of Science, University of Málaga,Málaga, Spain

Fatty acids and cholesterol compositions, in both fresh and mature cheeses, prepared inaccordance with the Manchego-type cheese with different fat compositions, using milkenriched with avocado oil were evaluated. Fresh cheese enriched with avocado oil showed anincrease of 52% in polyunsaturated acids in full-fat milk and 98% in skimmed milk. Maturemanchego style cheeses enriched with avocado oil showed an increase of 32% in polyun-saturated acids in full-fat milk and 61% in skimmed milk. Multivariate analysis (non-linearmapping, hierarchical cluster analysis, and linear discriminating analysis) of the fatty acidscomposition in newly prepared cheeses confirmed that they were different from the tradi-tional cheeses and that a small set of fatty acids (myristoleic, lauroleic, eicosenoic, caproleic,stearic, palmitoleic, and margaroleic) can be used to develop classification rules.

Keywords: Ewe cheese, Manchego cheese, Avocado oil, Fatty acids content, Cholesterolcontent.

[Supplementary materials are available for this article. Go to the publisher’s online editionof International Journal of Food Properties to view the free supplementary files.]

INTRODUCTION

The Mediterranean diet, based in olive oil, is generally believed to reduce the risk ofheart disease, Alzheimer’s disease, diabetes, and changes in the oxidative status of tissuescausing others diseases.[1–3] Extracted from the pericarp of the olive fruits, it is the majorsource of edible lipids that are consumed throughout the world.[4] Another important oilyfruit is the avocado (Persea americana Mill.), with the main European production of this

Received 6 March 2010; accepted 17 June 2010.Address correspondence to Manuel Algarra, Centro de Geología, Departamento de Geoelêneias,

Ambiento e Orden amento do Território do Porto, Faculdade de Ciênias, Universidade do Porto, Rua do CampoAlegre 687, Porto 4169-007, Portugal. E-mail: magonzal@fc.up.pt

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fruit located in the subtropical regions of Málaga and Granada, in southern Spain. Avocadooil, as a food substance, is used as an ingredient in recipes, as well as cooking oil.[5] Inaddition, it is used for lubrication[6] and is very sought after in cosmetics, where it is valuedfor its regenerative and moisturizing properties.[7–9] From the nutritional point of view,avocado is a high calorie fruit with a high unsaturated fatty acids content.[10] It is alsorich in vitamin E, ascorbic acid, vitamin B6, β-carotene, and potassium.[11–13] Thus, theindustrial production of functional foods based on avocado shows high potential.

Milk products play an important part in a healthy diet, as they contribute to intakesof essential nutrients and protein of high nutritional value. In Spain, dairy milk productscontribute more than 50% of the total calcium intake.[14] However, due to the high contentof cholesterol raising saturated fatty acids (SFA) in milk fat, a decrease in the intake offat-rich dairy products is recommended. Although there is no doubt that long-chain SFA inmilk fat increases plasma cholesterol,[15,16] there is some controversy regarding the specificeffect of milk products. Thus, dairy milk preparations were formulated by the milk industryto improve its natural composition.

This study presents the fatty acids composition and cholesterol contents of a partic-ular dairy prepared cheese, both fresh and mature (ripened for 4 months). It is one of themost popular varieties of cheese in Spain (known as ‘Manchego-type cheese’); it is madewith pure ewe milk from the machega breed (Ovis Aries) and is enriched with 2 g avocadooil/100 mL milk. The objective was to obtain a cheese with low cholesterol and saturatedfatty acids content and a high percentage of unsaturated fat by use of a milk prepara-tion based in avocado oil.[17] The fatty acids contents were subjected to a multivariateclassification (non-linear mapping, hierarchical cluster analysis, and linear discriminatinganalysis) to check for similarities and differences between the various samples and fattyacids compositions.

MATERIAL AND METHODS

Reagents and Materials

All the chemicals used were of the highest purity available, betulin (Lup-20(29) ene-3β, 28-diol, 99%); N, O-Bis (trimethylsilyl) trifluoroacetamide (≥98%), pyridine, standardfree fatty acids (Supelco® 37 component FAME Mix), and betulin (98%) were supplied bySigma-Aldrich Química (Barcelona, Spain). Reference standards for fatty acids were usedto quantify as methyl testers. Betulin (2.25 × 10-3 M) and KOH (2 M) were prepared inmethanol, respectively. Avocado oil was purchased from Laboratorie Soetenaey and Sictia(Fécamp, France).

Cheeses Manufactured and Storage

Cheeses were manufactured according to traditional Manchego type methods. Twotypes of natural milk are used. One with low fat milk (4.1%), obtained from ewes duringthree weeks subsequent to the birth of their lambs, to elaborate the Type I cheeses; and theother to elaborate Type II-F and Type III-F with a fat content of 6.5%. Raw ewe milk wasfiltered through a double membrane piece of porous paper that eliminated any possibleremnants, thus avoiding contamination by external germs and the consequent transmis-sion of undesirable flavours. It was then refrigerated at 4◦C. Three different ewe cheeseswere manufactured from unblended ewe milk in accordance with the following procedure:a commercial starter culture (Lactococcus lactis spp. lactis plus Lactococcus lactis spp.

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cremoris, from Degussa BioActives Deutschland GmbH & Co.KG, Freising, Germany)was added at the ratio of 2 g per 100 L of milk, and after 35 min at 30◦C was cut usinga 20-mm knife and stirred at 38◦C. The curd was molded and pressed for 2 h and saltedby immersion in 18% NaCl solution (pH 5.5) for 8 h at 14◦C and stored in a refrigeratedchamber at 4◦C. Cheeses from every treatment were weighed and chemically analysed.

Two different cheese types were prepared according to the raw milk fat content used.For each sample, two types of cheeses were made: one was analysed fresh, while the otherone was stored for 4 months to obtain the typical Manchego-type cheese and then analysed.In a preliminary test, several batches (B) of matured (M) cheeses were made to check thebehaviour of the starter preparations with the supplement of the avocado oil. The fattyacids, fat, and cholesterol content results of these preliminary samples were analysed andused in the chemometric classification (shown as supplementary information) where TypeI-M-B is made with low fat milk; Type II-M-B is made with full fat milk by means of amechanical procedure; and Type II-M-B∗ and Type II-M-B∗∗ are elaborated by differentmechanical procedures; according to this table, the incorporation of monounsaturated acidsare most efficient by mechanical procedure. As reference of them, was made the traditionalelaboration (see table in supplementary information for fatty matter, cholesterol, and fattyacids composition [Tables 1 and 2]).

After that a different set of cheeses were elaborated to analyze the compositionbefore the ripening, fresh (F), and after 4 months:

• Type I-F: Prepared with 4.5 L milk (fat matter content: 4.1%) enriched with 2 g avocadooil/100 mL milk), with a final fat content of 5.9%. After it was made, the final weightwas 742 ± 10 g. After ripening it is labeled as Type I-M.

Table 1 Fatty acids composition of the fresh cheese samples (%).

Fatty acid Type I-F Type II-F Type III-F

Butyric 0.00 0.00 0.00Caprylic 0.00 0.00 2.45Capric 7.39 6.62 7.33Lauric 3.72 3.53 4.02Myristic 8.89 8.83 10.62Palmitic 32.96 29.72 31.56Margaric 0.58 0.50 0.61Stearic 10.35 11.14 13.89Arachidic 0.52 0.40 0.32Behenic 0.17 0.15 0.19Lignoceric 0.05 0.05 0.05Caproleic 0.21 0.21 0.27Lauroleic 0.05 0.07 0.08Myristoleic 0.33 0.33 0.39Palmitoleic 3.85 2.58 1.09Margaroleic 0.22 0.21 0.24Oleic 23.59 30.21 23.28Linoleic 6.82 5.18 3.28Linolenic 0.00 0.00 0.00Eicosenoic 0.30 0.28 0.32

Type I-F: Fresh cheese made with 2 g avocado oil/100 mL milkwith low fat content; Type II-F: Fresh cheese made with 2 g avocadooil/100 mL milk with full fat content; Type III-F: Fresh cheese madewith full fat content (commercial cheese).

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Table 2 Fatty acid composition of the mature (M) cheese samples (%).

Fatty acid Type I-M Type II-M TMC

Butyric 3.14 4.20 2.36Caprylic 1.74 2.28 2.23Capric 5.22 6.78 6.97Lauric 2.68 3.53 3.74Myristic 6.75 8.67 10.23Palmitic 26.17 27.59 30.29Margaric 0.49 0.45 0.58Stearic 9.31 9.66 13.33Arachidic 0.38 0.40 0.61Behenic 0.15 0.12 0.17Lignoceric 0.06 0.04 0.05Caproleic 0.15 0.26 0.25Lauroleic 0.05 0.07 0.08Myristoleic 0.24 0.32 0.37Palmitoleic 2.88 2.40 1.05Margaroleic 0.20 0.19 0.23Oleic 34.19 27.99 23.63Linoleic 5.50 4.49 3.19Linolenic 0.42 0.34 0.32Eicosenoic 0.27 0.22 0.32

Type I-M: Mature cheese made with 2 g avocado oil/100 mL milkwith low fat content; Type II-M: Mature cheese made with 2 g avocadooil/100 mL milk with full fat content; Type III-M: Mature cheese madewith full fat content (commercial cheese).

• Type II-F: Prepared with 4.5 L full-fat milk (fat matter content: 6.5%) enriched with 2 gavocado oil/100 mL milk), presented a final fat content of the milk 8.1%. After that thecheese obtained weighed 960 ± 10 g. After ripening it is labeled as Type II-M.

• Type III-F: Finally, a batch of cheese made with full-fat milk (fat matter content of 6.5%)and used as reference, corresponding to commercial cheese. After ripening it is labeledas Type III-M.

Incorporation of Avocado Oil into Milk Fat-Globules

The implementation of avocado oil from embedded products, such as milk andcheese, required a homogeneous mixture of oil and milk. It was, therefore, necessarythat the molecules of oil were incorporated into the fat globules of milk. Thus, opticalmicroscopy was used to monitor the incorporation process of the avocado oil and the opti-mal concentration to obtain a homogeneous solution. Two methods of incorporation ofavocado oil into the fat globule were tested: stirring for 1 min at 1000 rpm and by ultra-sounds for 15 min at room temperature. In the case of the stirred procedure, two phases areobserved with up to 4% after 4 days. However, for milks under ultrasound and for all theconcentrations tested, two phases were observed with a low incorporation of avocado oil.Thus, the process used was mechanical stirring.

Determination of Total Fatty Matter

Respective samples of cheeses (3 g) were heated at 60◦C with 10 mL of concentratedHCl (10 M). After that, three extractions with n-hexane were made with 2 × 20 mL and 1

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× 10 mL, and when solvent evaporation was carried out, the amount of fatty matter wasdetermined by weight.[18]

Cholesterol Analysis

Five hundred grams of cheese, finely chopped and crushed, were extracted withn-hexane for 10 min at room temperature; after that 5 g of the extract were treated undergentle reflux, with KOH (6.5 g), methanol (40 mL), H2O (10 mL), and betulin (1 mL),used as standard. Previous to cooling at room temperature, H2O (100 mL) was added andextracted with diethyl ether (2 × 50 mL). The resulting organic mixture was washed withH2O (3 × 50 mL) and the organic layer was washed with (20 mL) of brine. It was then driedwith Na2SO4 and filtrated; the solvent was removed using a rotary evaporator system. Theunsaponifiable residue was dissolved in a 1:1 hexane:ether solution that afforded the cor-responding sterols, separated by using a silica column as the mobile phase of 1:1 hexane:ether solution. The sterols fraction obtained were concentrated under vacuum and silanizedby 30 µL of N, O-Bis (trimethylsilyl) trifluoroacetamide in 60 µL of pyridine solution for30 min.

Fatty Acids Analysis

The fatty acids composition was determined after methylation.[18] Cheese extract(0.5 g) was vigorously stirred for 5 min with 0.5 g of KOH/methanol solution and n-hexane (10 mL) and left to stabilize. The organic phase was injected and the fatty acididentification was done by comparing its retention time with the standard mixture. Thefinal concentrations (%) of fatty acids were reported as the percentage of total FFAs. Allanalyses were performed in duplicate, and the analysis was repeated if necessary.

Apparatus

Analysis was performed using a Clarus 500 instrument (PerkinElmerm, Norwalk,CT, USA), with the use of gas chromatography analyses equipped with a flame ioniza-tion detector (GC-FID) and an auto sampler system. Data acquisition and processing wereperformed using the Soft-link and Total-Chrom Software application (PerkinElmer®’sTotalChem® Chromatography Data Systems [CDS] Software). For cholesterol analysis,3 µL of standard/sample solution was injected, via the autosampler, to a fused silica capil-lary column (25 m × 0.25 mm i.d.) coated with 5% phenyl and 95% dimethylpolysiloxane(Varian CP-Sil 8CB, Varian, Dallas, TX, USA). The injector operating conditions wereas follows: injector temperature was 260◦C, hydrogen carrier gas flow maintained at1 mL/min. The oven temperature was adjusted to 300◦C and the detector was at 310◦C.For free fatty acids analysis, 1 μL of standard/sample solution was injected, via theautosampler, to a fused silica capillary column (60 m × 0.25 mm i.d.) coated with 90%polybiscyannopropyl and 10% cyanopropylphenylsiloxane (Supelco SP-2380, Sigma-Aldrich Química, Barcelona, Spain). The injector operating conditions were as follows:injector temperature was 250◦C, hydrogen gas flow was maintained at 1 mL/min. Theoven and the detector temperature were adjusted to 180 and 260◦C, respectively. ThepH was measured by an XS 100 Eutech (Thermo Scientific, Surrey, UK). The opticalimages were recorded by means of a Nikon Microscopy Eclipse E 800, connected to a

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Nikon DXM1200 digital camera, controlled by a PC using the ACT-1 software (NikonCorporation, Sendai, Japan).

Multivariate Chemometric Analysis

A chemometric classification of the cheeses was done using firstly a non-supervisedapproach [non-linear mapping (NLM) and hierarchical clustering analysis (HCA)] fol-lowed by a brief supervised analysis (linear discriminate analysis). Non-linear mapping(ALSCSAL) and HCA were used as an unsupervised exploratory technique to detect nat-ural similarities among the fatty acids content of the cheeses. Squared Euclidean distancewas always used as the similarity index for clustering, preferably using the average linkageand centroid method. Raw data was standardized based on variable Z-scores. The discrimi-nate capacity power of the variables was assessed with linear discriminate analysis. All thestatistical multivariate analysis was performed using the SPSS 16.0 for Windows software(SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

Figure 1 shows the optical microscopy images of the different dairy preparationsaccording to the selected procedure of incorporating the avocado oil into the milk. Theseexperiments were carried out using different ranges of oil concentration, from 0–10%(v/v), and left for several days to observe the evolution. Figures 1a and 1b (1 and 2%added avocado oil) show that oil is found on the surface of the fat globules (dark spots).In the case of milks with 4% (Fig. 1c) and 6% (Fig. 1d) avocado oil is present on the surface

(a)

100 µm 100 µm

(b)

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(d)

Figure 1 Optic microscopy of mixed avocado oil-milk at (a) 1%, (b) 2%, (c) 4%, and (d) 6%. (Color figureavailable online.)

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of the fat globule as micro-vesicles. These observations show that cheese preparations witha maximum of 2% avocado oil can be prepared and are analysed in the following sections.

Fat Content, Fatty Acids, and Cholesterol Composition

in the Fresh Cheeses

Fat content is the most quantitatively and qualitatively variable component ofcheeses, depending on the lactation stage, season, breed, genotype, and feeding of theanimal. This last important factor has been studied in depth and the main findings con-cerning the impact of feeding, basic roughage, and lipid supplementation, on quantitativeand qualitative variation.[19] The fat content obtained for the fresh cheeses were Type I-F:28.07%; Type II-F: 33.61%; and Type III-F: 27.21%.

The detailed fatty acids composition of fresh cheese dairy products is shown inTable 1. Saturated fatty acids (SFAs) were predominant, 71.04% for Type III-F; 60.94 and64.63% for Type II-F and Type I-F, respectively. Monounsaturated fatty acids (MUFAs)reached a value of 25.35% for Type III-F; 33.61 and 28.25% for Type II-F and Type I-F,respectively. Palmitic and stearic acids were the major SFAs present in both. Oleic acidand palmitoleic acid were the most abundant MUFAs, where the total content in oleic acidwas 23.28% for Type III-F; 30.21 and 23.59% for Type II-F and Type I-F, respectively.Previous studies have reported that the concentrations of unsaturated fatty acids increaseand those of saturated fatty acids decrease.[20]

The analysis of polyunsaturated fatty acids (PUFAs) showed a composition of 3.60%for Type III-F; 5.46 and 7.12% for Type II-F and Type I-F, respectively. In the present study,the PUFAs’ concentration increased with the content obtained for Type III-F at 51.7 and97.8% for Type II-F and Type I-F, respectively. The cholesterol contents were measured as69.36 mg per 100 g of sample for the Type III-F cheese; 56.40 and 63.72 mg per 100 mgof sample for Type II-F and Type I-F, respectively. Therefore, the hand-made fresh cheesewith an added 2% of avocado oil represents a good source of PUFAs for humans with lowerconsumption of other lipids. Figure 2 shows a bar illustration of the results previouslydiscussed.

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Type I-F

SFAs (%)

MFAs (%)

PFAs (%)

Fat (%)

mg Cholesterol / 100 g cheese

Figure 2 Total content of different fatty acids, fat, and cholesterol of the fresh cheeses.

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Fat Content, Fatty Acids, and Cholesterol Composition

of the Mature Cheeses

Figure 3 summarises the global composition of fatty acids, fat content, and choles-terol contents of the previous dairy products, after the typical La Mancha cheese varietyhad been left in the ripening process for 4 months. The fat content in theses cheeses were43.0%, an increase of 58% in respect to the fresh Type III-F cheese and 45.3 and 45.0%where the fat increases were of 34.8 and 60.3% for Type II-M and Type I-M, respectively.

The percentage values of the (SFAs) were predominantly 70.56% for Type III-M and63.72 and 56.09% for Type II-M and Type I-M, respectively, where the most abundant fattyacids were palmitic, stearic, and myristic acids. Monounsaturated fatty acids (MUFAs)reached a value of 25.61% for Type III-M; 31.23 and 37.71% for Type II-M and TypeI-M, respectively. Oleic acid corresponds to more than 95% of MUFAs in all cheeses.The analysis of PUFAs afforded a composition of 3.83% for Type III-M; 5.05 and 6.19%for Type II-M and Type I-M, respectively. In the present study, the concentration of PUFAsrepresents an increase when compared to the value obtained for the Type III-M of 31.85 and61.62 % for Type II-M and Type I-M, respectively.

The cholesterol contents were measured as 91.54 mg/100 mg of sample for TypeIII-M; 71.07 and 62.41% for Type II-M and Type I-M, respectively. Therefore, the hand-made Manchego type cheese enriched with 2% (v/v) avocado oil represents a good sourceof PUFAs for humans with lower consumption of other lipids. Figure 3 shows a barillustration of the results previously discussed.

Multivariate Chemometric Analysis

Figure 4 shows the NLM of the cheese data set. The analysis of this figure shows thatthe Manchego cheese is quite dissimilar from the other cheeses and it shows the existenceof some clusters of similar cheeses. However, a detailed analysis of the NLM should be

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Type II-M Type III-M

Type I-M

SFAs (%)

MFAs (%)

PFAs (%)

Fat (%)

mg Cholesterol / 100 g cheese

Figure 3 Total content of different fatty acids, fat, and cholesterol of the ripened cheese samples.

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Figure 4 Nonlinear map (Stress = 0.04832 and RSQ = 0.98523) of the cheeses with the classes detected byhierarchical cluster analysis highlighted.

Figure 5 Dendograms obtained using the centroid (a) and average linkage (b) methods of the hierarchical clusteranalysis of the cheese data set. 4.0 Reference; 4.0 Type I-M-B; 3.0 Type II-M-B∗; 3.0 Type II-M-B∗∗; 2.0 TypeIII-M; 2.0 Type III-F; 1.0 EC; 1.0 Type I-M; 1.0 Type II-F; 1.0 Type I-F.

done with caution because it is a bi-dimensional projection of the real hyperspace graphrepresented by the 20 fatty acid concentrations. Indeed, the error function (Young’s S-stressformula) of the NLM of Figure 4 is 0.04832. Although the NLM suggests some similaritiesamong the fatty acids compositions of the cheeses, a more rigorous analysis of the clustercomposition should be done with HCA. Figure 5 shows two dendograms of the cheese dataset. Dendogram analysis shows that some cheeses cluster at distances close to zero, and at

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a rescaled distance of about ten, three clusters and two outlier cheeses (Reference and TypeI-M-B) are detected. Table 3 presents the average and standard deviation of the fatty acidsof the detected classes. The borders represented in the NLM of Figure 4 highlights theclusters of similar cheeses and outlier cheeses.

The three clusters detected by NLM and HCA correspond to fresh and maturecheeses with added advocado oil (Type II-M, Type-I-M, Type II-F, and Type I-F), freshand mature batch chesses (Type II-M and Type III-F), and preliminary cheese prepara-tions with whole milk (Type II-M-B∗ and Type II-M-B∗∗). The detected outlier, Referenceand Type I-F-B, correspond respectively, to the traditional cheese and to a prelimi-nary cheese preparation with skimmed milk enriched with 2% avocado oil. This resultshows, as expected, that the preliminary cheese preparations are different from the othercheeses. Also, and as expected, the reference cheeses (Type III-F and Type II-M) aredifferent from the cheeses with 2% avocado oil (Type II-M, Type I-M, Type II-F, andType I-F).

A detailed analysis of the dendograms of Figure 5 shows that by increasing thesimilarity, for example to a rescaled distance cluster combination of 5, discriminationbetween the cheeses with 2% avocado oil is achieved. Indeed, now two sub-clusters areobserved (Type II-M and Type I-M) and (Type III-F and Type I-F), and they correspondrespectively to cheeses prepared from milk with 6.5 and 4.1% fat content. In order to assess

Table 3 Average and standard deviation of the fatty acids of the classes of cheese detected by hierarchical clusteranalysis.

Class 1(Type II-M, Type I-M,Type II-F, Type I-F)

Class 2(Type III-F,Type III-M)

Class 3(Type II-B∗,Type II-B∗∗)

Fatty acid Average Standard dev. Average Standard dev. Average Standard dev.

Butyric 1.83 2.16 1.18 1.67 1.25 1.77Caprylic 1.00 1.18 2.34 0.15 2.51 0.30Capric 6.50 0.91 7.15 0.25 0.90 1.07Lauric 3.36 0.46 3.88 0.19 4.30 0.60Myristic 8.28 1.02 10.42 0.27 9.57 1.31Palmitic 29.11 2.95 30.92 0.89 26.42 1.33Margaric 0.50 0.05 0.59 0.02 0.55 0.08Stearic 10.11 0.80 13.61 0.39 9.79 1.29Arachidic 0.42 0.06 0.46 0.20 0.25 0.01Behenic 0.14 0.02 0.18 0.01 0.13 0.01Lignoceric 0.05 0.00 0.05 0.00 0.05 0.00Caproleic 0.20 0.04 0.26 0.01 0.00 .00Lauroleic 0.06 0.01 0.08 0.00 0.00 0.00Myristoleic 0.30 0.04 0.38 0.01 0.00 0.00Palmitoleic 2.92 0.64 1.07 0.02 2.05 0.71Margaroleic 0.20 0.01 0.23 0.01 0.20 0.01Oleic 28.99 4.42 23.45 0.25 28.83 4.34Linoleic 5.49 0.97 3.23 0.06 4.66 0.93Linolenic 0.19 0.22 0.16 0.23 0.53 0.04Eicosenoic 0.26 0.03 0.32 0.00 0.04 0.05

Type I-F-B: Fresh cheese made with 2 g avocado oil/100 mL milk with low fat content; Type II-F-B∗: Freshcheese made with 2 g avocado oil/100 mL milk with full fat content and elaborated by mechanical procedures;Type II-F-B∗∗: Fresh cheese made with 2 g avocado oil/100 mL full fat content.

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Table 4 Wilks’ Lambda and F tests of group means forthe linear discriminate analysis of cheese classes.

Fatty acid Wilks’ lambda F

Butyric 0.963 0.096Caprylic 0.513 2.369Capric 0.583 1.791Lauric 0.459 2.950Myristic 0.429 3.333Palmitic 0.581 1.800Margaric 0.575 1.851Stearic 0.161 12.995Arachidic 0.496 2.536Behenic 0.407 3.648Lignoceric 1.000 0.000Caproleic 0.073 31.718Lauroleic 0.053 45.000Myristoleic 0.033 72.394Palmitoleic 0.272 6.703Margaroleic 0.308 5.625Oleic 0.632 1.455Linoleic 0.353 4.586Linolenic 0.520 2.305Eicosenoic 0.057 41.108

the discriminating capacity of the fatty acids among the three classes that compose thedata set, the Wilks’ Lambda and F-tests were performed (Table 4). The smallest value ofWilks’ Lamda and the highest value of the F-tests depict the highest discriminating valueof the variable.[21] The fatty acids with the higher discriminating capacity are myristoleic,lauroleic, eicosenoic, caproleic, stearic, palmitoleic, and margaroleic. Consequently, clas-sification strategies based on the fatty acids composition for these cheeses should focus onthese substances.

CONCLUSIONS

The results presented and discussed in this study showed that cheeses obtainedfrom ewe milk supplemented with avocado oil at 2% (v/v), contained a smaller per-centage of SFAs and a higher percentage of MUFAs and PUFAs, compared with thetraditional Manchego type cheeses. Also, these new chesses present lower cholesterol con-tents. Multivariate analysis of the fatty acid composition in the newly prepared cheesesenriched with 2% avocado oils confirms that they are different from the traditional cheesesand that a small set of fatty acids can be used to develop classification rules. Thus, dueto the SFAs, MUFAs, PUFAs, and cholesterol composition, these new cheeses can help toprevent excessive fat intake related diseases. Accordingly, the fat content in comparisonwith other cheeses type varieties, such as Camembert (average of 48 g/100 g cheese),[22]

Cheddar (48 g/100 g cheese),[23] Manchego type cheese (average of 54 g/100 g cheese,[24]

or 30 g/100 g cheese,[25] Roncal type (average of 48 g/100 g cheese,[26] and Swiss cheese(30 g/100 g cheese),[27] offer a good alternative to traditional and commercials, where theincorporation of avocado oil does not increase this chemical parameter.

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ACKNOWLEDGMENTS

The authors would like to thank Dehesa Los Llanos (Albacete, Spain) for the purchase of theewe milk, industry facilities for making the cheeses, and Salsa Natura (Málaga, Spain) for fundingthis project. M. A. would like to thank Fundação para a Ciência e a Tecnologia (Lisboa, Portugal),under the frame of the Ciência 2007 program.

REFERENCES

1. Hall, J.H.; Moore, S.; Harper, S.B.; Lynch, J.W. Global variability in fruit and vegetableconsumption. American Journal Prevention Medicine 2009, 36(5), 402–409.e5.

2. Day, J.; Jones, D.P.; Goldberg, J.; Ziegler, R.; Bostick, R.M.; Wilson, P.W.; Manatuga, A. K.;Shallenberger, L.; Jones, L.; Vaccarino, V. Association between adherence to the Mediterraneandiet and oxidative stress. American Journal Clinical Nutrition 2008, 88 (5), 1364–1370.

3. Hu, F.B.; Manson, J.E.; Willet, W.C. Types of dietary fat and risk of coronary heart disease:Critical review. Journal of the American Colleague of Nutrition 2001, 20 (1), 5–19.

4. Esteves da Silva, J.C.G. Chemometric classification of cultivars of olives: Perspectives onPortuguese olives. In: Olives and Olive Oil in Health and Disease Prevention; Preedy, V.R.;Watson, R.R.; Eds.; Academic Press (Elsevier), 2010, Chap. 4, pp. 33–42.

5. Saura-Calixto, F.; Goñi, I. Antioxidant capacity of the Spanish Mediterranean diet. FoodChemistry 2006, 94, 442–447.

6. Tennant, D.R. The usage, occurrence and dietary intakes of white mineral oils and waxes inEurope. Food Chemistry Toxicology 2004, 42, 481–492.

7. Cole, E.T.; Cadé, D.; Benameur, H. Challenges and opportunities in the encapsulation of liq-uid and semi-solid formulations into capsules for oral administration. Advanced Drug DeliveryReview 2008, 60, 747–756.

8. Boileau, C.; Martel-Pelletier, J.; Caron, J.; Cheng, S.; Pelletier, J.P. Protective effects of totalfraction avocado/soybean unsaponifiables (ASU) on the structural changes in experimental dogosteoarthritis: Inhibition of nitric oxide synthase and MMP-13. Arthritis Research Therapeutic2009, R41.

9. Benaiges, A.; Guillén, P. Botanical extracts. Actives for skin-care products. Actives for personalhygiene and other toiletry products. Actives with specific claims. Analysis of Cosmetic Products2007, 345–363.

10. Moreno, A.O.; Dorantes, L.; Galindez, J.; Guzman, R.I. Effect of different extraction methodson fatty acids, volatile compounds, and physical and chemical properties of avocado (PerseaAmericana Mill.) oil. Journal Agriculture Food Chemistry 2003, 51, 2216–2221.

11. Bergh, B. Nutritious value of avocado. California Avocado Society Yearbook 1992, 76, 123–135.12. Gains, A. Avocados will lower your cholesterol. Here is Health 1992, 36–37.13. Lassen, S.; Bacon, K.; Sutherland, J. Vitamin D and pro-vitamin D content of the avocado.

Journal Food Science 2006, 10, 1–4.14. Orozco, P.L.; Zwart, M.S.; Garrofa, E.G.; Domínguez, C.O. Prediction of the total calcium

intake from consumption of milk products in Spain adult population. INDICAD Study. AtenciónPrimaria 2004, 33, 237–243.

15. Tholstrup, T.; Høy, C.E.; Andersen, L.N.; Christensen, R.D.K.; Sandström, B. Does fat inmilk, butter and cheese affect blood lipids and cholesterol differently?. Journal of the AmericanCollege of Nutrition 2004, 23, 169–176.

16. Thompson, L.U.; Jenkins, D.J.A.; Amer, M.A.V.; Reichert, R.; Jenkins, A.; Kamulsky, J. Theeffect of fermented and unfermented milks on serum cholesterol. American Journal ClinicalNutrition 1982, 36, 1106–1111.

17. Cabello, R.M.; Vadillo, J.M.; Casado, J.; Jiménez-Jiménez, J. Milk preparation based in avocadooil. Dehesa de los Llanos S.L. Patent ES 2, 301–365; 2008.

Dow

nloa

ded

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rsity

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aga]

at 0

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201

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808 ALGARRA ET AL.

18. International Dairy Federation. Milk fat. Preparation of fatty acid methyl esters. IDF StandardISO 15884-IDF 182. International Dairy Federation: Brussels, Belgium, 2002.

19. Sanz-Sampelayo, M.R.; Chilliard, Y.; Schmidely, P.; Boza, J. Influence of type of diet on the fatconstituents of goat and ewe milk. Small Ruminant Research 2007, 68, 42–63.

20. Martinez, N.L.; Moreno, R.V. Parameters for determining the maturity of avocados. Industries.Alimentaires et Agricoles 1995, 112, 200–203.

21. Pinheiro, P.B.M.; Esteves da Silva, J.C.G. Detection of 2,4,6-trichloroanisole in chlorinatedwater at nanogram per litre levels by SPME-GC-ECD. Analytical and Bioanalytical Chemistry2005, 382, 341–346.

22. Guizani, N.; Kasapis, S.; Al-Attabi, Z.H.; Al-Ruzeiki, M.H. Microbiological, physicochemical,and biochemical changes during ripening of camembert cheese made of pasteurized cow’s milk.International Journal of Food Properties 2002, 5, 483–494.

23. Ereifej, K.I. Characteristics of milk, cheddar-like and white cheese obtained from three goatphenotypes during three lactation periods. International Journal of Food Properties 2006, 9,803–811.

24. Cabezas, L.; Sánchez, I.; Poveda, J.M.; Seseña. S.; Palop, M.L. Comparison of microflora, chem-ical and sensory characteristics of artisanal Manchego cheeses from two dairies. Food Control2007, 18, 11–17.

25. Illescas-Chavez, E.; Vélez-Ruiz, J.F. Effect of the salting process on the mass transfer kineticsof manchego type cheese. International Journal of Food Properties 2009, 12, 791–807.

26. Taborda, G.; Molina, E.; Martínez-Castro, I.; Ramos, M.; Amigo, L. Composition of the water-soluble fraction of different cheeses. Journal Agricultural Food Chemistry 2003, 51, 270–276.

27. Kocaoglu-Vurma, N.A.; Harper W.J.; Drake, M.A.; Courtney, P.D. Microbiological, chemical,and sensory characteristics of Swiss cheese manufactured with adjunct lactobacillus strainsusing a low cooking temperature. Journal Dairy Science 2008, 91, 2947–2959.

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