1. Introduction
Although dairy products in general have the image of being healthy foods [
1], this image is often not perceived for products with a high fat content such as cream, butter, and cheeses due to the high amount of cholesterol and saturated fatty acids, mainly in mature cheeses with high fat content, and consequently, the reduction of cholesterol in these products and especially in matured cheeses would reduce the risk of cardiovascular diseases.
To reduce the risk of coronary heart disease, the World Health Organization and the American Heart Association advise consumers to consume fewer saturated fatty acids and cholesterol. A market for low cholesterol products has been generated by this advise and radical view points [
2]. Dairy products with lower cholesterol is becoming more popular today. The removal of cholesterol from food can be accomplished using a variety of techniques, such as the incorporation of vegetable oils [
3,
4], distillation and crystallization [
5,
6], adsorption with saponin and digitonin [
7,
8], assimilation of cholesterol by microorganism enzymes [
9,
10], and removal by supercritical carbon
Dioxide extraction [
11,
12]. Several investigations have been detailing the usage of β-CD in food have been reported [
13,
14,
15]. It has been demonstrated that β-CD molecules can be utilized to eliminate cholesterol from milk and dairy products in an efficient manner while being non-toxic and indigestible [
15]. Seven glucose units make up the cyclic oligosaccharide known as β-CD, which is produced from starch by the enzyme cyclodextrin glycotransferase, which also breaks the polysaccharides chain to generate cyclic polysaccharide molecules.
The doughnut-shaped molecule of β-CD has a circular hydrophobic region in the center that is about the same size as a molecule of cholesterol. This gives the molecule an affinity for non-polar molecules like cholesterol. The cavity’s radius nearly perfectly fits a allowing the molecule to have a preference for non-polar compounds like cholesterol The cavity’s radius almost perfectly fits a cholesterol molecule, giving β-CD a high specificity for forming a 2:1 host/guest inclusion insoluble complex with cholesterol that can be separated by centrifugation or sedimentation [
16,
17].
One of Spain’s most recognizable hard cheeses is Manchego. It is produced by a designation of origin in the Castilla la Mancha region of Spain using only pure sheep’s milk [
18]. Rich in fat (fat content in dry cheese is greater than 50%), Manchego has a distinctively strong flavor that develops over time. Although studies have been conducted to show that β-CD is a great ingredient for removing cholesterol from Manchego cheese [
18], the potential effect of curd washing on this process has not yet been investigated. In the literature, we found works in which curd washing was used to control the lactose ratio in cheese making [
19] or the insoluble Ca content [
20]. However, little information is available on the effect of such curd washing on the chemical and organoleptic properties of cheese [
21,
22], and there are no data available in the particular case of β-CD-treated milk used in the manufacture of low-cholesterol cheeses.
The present study, which was a continuation of a previous research by Alonso et al. [
18], aimed to assess the effect of the technological process of curd washing on the final quality of β-CD-treated Manchego cheese as well as on the reduction of residual β-CD throughout the manufacturing process of low-cholesterol Manchego cheese.
3. Materials and Methods
3.1. Chemicals
α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and all reagents were supplied by Sigma (St. Louis, MO, USA). Deionized water was prepared by a water purification system (Millipore Co., Burlington, MA, USA).
3.2. Manchego Manufacture
An amount of 100 L of pasteurized ewe’s milk from Monte de Toledo (Toledo, Castilla la Mancha, Spain) (15 s at 72 °C) containing 1% of β-CD (
wt/
vol) was placed in a cold room at 4 °C and mixed by stirrer for 30 min. After mixing, the treated milk was left static overnight at 4 °C for binding the cholesterol to the β-CD. A cholesterol-β-CD complex was formed and was sedimented by precipitation [
15].
Manchego cheese was manufactured with pasteurized ewe’s milk (15 s at 72 °C) in two experiments, each one performed in triplicate. In each experiment, 100 L of pasteurized refrigerated whole milk was treated with/without BCD. Milk at 30 °C was inoculated at rate of 1% (
wt/
wt) of commercial mixed of
L. lactis strains. After 20 min, 5 mL of rennet (Maxiren 1:15,000 strength
vol/
vol; Gist Brocades, Deft, The Netherland) was added to each vat of milk, renneting the milk in 40 min. The washed control cheese (WCC) without β-CD and the washed experimental cheese (WEC) with 1% of β-CD in the milk were made by washing the curd with deionized water twice. The gel was cut into 10 mm cubes, left in the curd/whey mixture for 15 min, and then stirred at 37 °C for 10 min. Stirring was then stopped, and the whey was removed and replaced by the same volume of deionized water at 37 °C. The curd–water mixture was again stirred for 15 min at 37 °C and drained off. The cheeses were pressed for 20 h at 20 °C into molds, brine salted at 12 °C for 24 h, and ripened for three months at 14 °C and 85–90% relative humidity [
32].
3.3. Gross Composition
The fat, moisture, pH, and protein contents and nitrogen fractions were determined using the method in [
33].
3.4. Beta Cyclodextrin Analysis
The Alonso 2008 method [
34] was used to analyze CD. The internal standard for quantitative analysis, 5 mg of β-CD dissolved in 1 mL of water, was added to a sample of 10 g of cheese. It was shaken for two minutes at 40 °C, centrifuged for thirty minutes at ambient temperature at 40,000 rpm to separate the upper layer, and then the upper layer was filtered through a 0.45 μm Millipore Co. membrane. The internal standard-spiked supernatant was delivered to the autosampler in a 30 μL aliquot. 10 μL aliquot of the supernatant was injected for high performance liquid chromatography (HPLC) analysis.
The Waters Empower 2 chromatography data program (Waters, Milford, MA, USA) was used for data collecting and analysis during the HPLC analysis using a Waters Alliance 2695 separation module connected to a 410 refractive index (RI) detector. On a YMC ODS AQ column (Teknochroma, Barcelona, Spain), separation was done. Methanol and water (7:93) in an isocratic environment and at a flow rate of 1 mL/min made up the mobile phase composition. The internal standard was used to prepare the standard solutions in water to determine the elution time, and the sample peak area of β-CD was compared to the internal standard to determine the β-CD’s quantity.
3.5. Lipid Extraction
A process described by an International Standard Method for Milk and Milk Method was used to extract lipids from samples [
35]. It involved adding an ammonia ethanol solution to a test portion and then extracting the lipids with diethyl ether and hexane. The solvent then entirely evaporated and the top layer was removed. The lipid extracts were collected, put into amber glass vials, flushed with nitrogen, and kept at 20 °C until analysis.
3.6. Determination of Cholesterol
The method used for cholesterol analysis was with capillary gas chromatography (GC) with direct injection of milk fat as described by Alonso 1995 [
36]. About 30 mg of anhydrous milk fat were dissolved in 1 mL of hexane together with 0.1 mL of 5 cholestane as the internal standard (3.5 mg/mL in hexane) to be used in GC analysis. The Agilent Technology 6890 chromatograph (Palo Alto, CA, USA) was used for the GC analysis for free cholesterol using direct injection with flame ionization detector. An HP 5 fused silica capillary column (30 m × 0.32 mm i.d. 0.25 mm thickness) was employed for the analyses.
3.7. Fatty Acids and Triglycerides Analysis
Fatty acid methyl esters (FAMES) were prepared by alkaline catalyzed methanolysis of the extracted lipids using 2 N KOH in methanol. An Agilent Technology 6890 chromatograph (Palo Alto, CA, USA) equipped with a FID detector, were used for analysis. The technique reported by Alonso et al., 1999 [
37] was utilized to separate fatty acids using a CP Sil 88 fused silica capillary column (50 m × 0.25 mm i.d. 0.2 m film thickness, Chrompack, CA, USA). The technique described by Alonso et al., 1999 [
37] was utilized to separate fatty acids using a CP-Sil 88 fused-silica capillary column (50 m × 0.25 mm i.d. 0.2 m film thickness, Chrompack, CA, USA). A flame ionization detector-equipped Agilent gas chromatograph 6890 (Palo Alto, CA, USA) was used for the GC analysis of triglycerides. The method described by Alonso 1993 [
38] was utilized to undertake the analyses using a WCOT fused silica capillary column (25 m × 0.25 mm 0.1 m film thickness) coated with OV 17 TRI.
3.8. Phospholipid Analysis
Using 2 g of freeze-dried cheese sample, 2 g of sea sand, and loading a stainless steel extraction cell coated with filters on both sides, cheese fat was extracted using an Accelerated Solid Extraction ASE 200 extractor (Dionex Corp., Sunnyvale, CA, USA). Dichloromethane methanol solution (2:1,
vol/
vol) was used as the solvent mixture and 10.3 MPa of pressure was a fixed condition during the extraction to provide the highest cheese fat yield possible [
39].
The separation of lipid classes was accomplished in an HPLC system (model 1260; Agilent Technologies Inc.) coupled with an evaporative light scattering detector (SEDEX 85 model; Sedere SAS, Alfortville Cedex, France) using the method [
39].
3.9. Analysis of Volatile Compounds
The analysis of the volatile fraction was performed by headspace gas chromatography-mass spectrometry (GC-MS) described by Alonso et al., 1999 [
40].
3.10. Short-Chain Free Fatty Acids
Cheese samples (1 g) were homogenized in 20 mL of distilled water, centrifuged at 10,000 rpm for 10 min, and filtered through a 0.40 μm filter to determine short chain free fatty acids (SCFFAs). Analyses were performed using a Hewlett Packard model 5890 A instrument with a flame ionization detector on a capillary silica column (HP FFAP, 30 m × 0.25 mm ID, 0.25 μm film thickness, Agilent J & W). Each peak area of a specific FFA was compared to the peak area of 2 ethyl butanoic acid as an internal standard for the quantitative analysis.
3.11. Sensory Analysis
Twenty-two trained sensory panelists evaluated randomly coded cheeses. The flavor, aroma, color, texture, and acceptability were evaluated on a five-point scale (1 = poor, 5 = excellent).
3.12. Statistical Analysis
Analysis of variance (ANOVA) was applied to the experimental data using SAS statistical software (version 8.02, SAS Institute Inc., Cary, NC, USA). A Student’s t test was used for statistical analysis, and a (p ≤ 0.05) was used to indicate statistical significance.
4. Conclusions
The aim of this study was to evaluate the effect of curd washing on the reduction in cholesterol induced by β-CD in pasteurized ewe’s milk Manchego cheese, considering the main components of milk, lipids, and flavor characteristics in regular Manchego cheese. An approximately 98.45% cholesterol reduction was observed in the washed experimental cheeses that were treated with β-CD. The remaining residual β-CD from the effect of curd washing was 0.15%. The chemical properties (fat, moisture, and protein) did not change as a result of curd washing with or without β-CD. The amounts of different components of the lipid fraction (fatty acids, triglycerides, and phospholipids) were similar in both the treated and the untreated cheese as a result of curd washing and with or without β-CD. The β-CD had no effect on the majority of flavor components and short chain free fatty acids. When curd washed with or without CD, there were no differences in the sensory qualities (flavor, aroma, color, texture, and acceptability). The β-CD molecules are edible and non-toxic, they can be employed safely in the cheese manufacturing process to remove cholesterol, which results in an 85% reduction in residual β-CD after curd washing. Therefore, the results of the current investigation revealed that removing cholesterol from Manchego cheese while maintaining its chemical qualities may be accomplished by treating β-D with the effect of curd washing improving the reduction in residual β-CD.