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A novel functional full-fat hard cheese containing liposomal nanoencapsulated green tea catechins: manufacture and recovery following simulated digestion Ali Rashidinejad,*a,b E. John Bircha and David W. Everett†b (+)-Catechin or green tea extract were encapsulated in soy lecithin nanoliposomes and incorporated into a full-fat cheese, then ripened at 8 °C for 90 days. Cheese samples were subjected to simulated gastrointestinal digestion to measure total phenolic content (TPC) and antioxidant activity of the cheese digesta, and to determine the catechin recovery after digestion by high performance liquid chromatography (HPLC). Addition of catechin or green tea extract significantly (P ≤ 0.05) increased TPC and antioxidant activity (measured by ferric reducing antioxidant power and oxygen radical absorbance capacity) of the full-fat cheese without affecting pH or proximate composition. HPLC analysis confirmed retention of encapsulated catechins in the cheese curd; however, individual catechins were recovered in differing

Received 15th March 2016, Accepted 25th June 2016

amounts (15–52%) from cheese digesta after 6 h of digestion. Transmission electron microscopy and

DOI: 10.1039/c6fo00354k

Fourier transform infrared spectroscopy provided evidence for association of nanoliposomes with the surface of milk fat globules inside the cheese matrix. The study shows the potential for using cheese as a

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delivery vehicle for green tea antioxidants.

1.

Introduction

Green tea catechins are well-recognised for having a wide range of health benefits, including anti-carcinogenic properties,1,2 anti-cardiovascular disease potential,3 anti-diabetes benefit,4 HIV protection,5 and anti-senescence.5 Green tea antioxidants are also used for increasing the stability and shelf-life of food6 by delaying lipid oxidation in food.7 The in vitro antioxidant activity of tea catechins enables scavenging of reactive oxygen and nitrogen species, chelation of redox-active transition metal ions, inhibition of redox-sensitive transcription factors, and prevention of pro-oxidant enzyme activity.7,8 For these reasons, there is increasing interest in the application of tea antioxidants (catechins) in dairy products.9–13 There is concern about the cost of these pure compounds when a functional food is made on an industrial scale. Hence, incorporating an extract from green tea, containing catechins, is a less expensive, but potent source of antioxidants for incorporation into food matrices, such as cheese, to create new functional foods.

a

Department of Food Science, University of Otago, PO Box 56, Dunedin 9054, New Zealand. E-mail: [email protected]; Fax: +64 3 479 7567; Tel: +64 3 479 7545 b Riddet Institute, Private Bag 11 222, Palmerston North 4442, New Zealand † Present address: Dairy Innovation Institute, California Polytechnic State University, San Luis Obispo, CA 93407, USA.

This journal is © The Royal Society of Chemistry 2016

Cheese is a good candidate for the delivery of green tea antioxidants because of the intrinsic nutritional value, variety for many taste preferences, and long shelf-life. However, there are considerable technical challenges to be overcome. Previously, we reported on the incorporation of different concentrations (125–1000 ppm) of the free form of (+)-catechin from green tea into low-fat cheese.14 The results showed that, although catechin was able to increase the antioxidant activity and phenolic properties of cheese, the increase was not proportional when the concentration of the phenolic compounds was doubled (P > 0.05), probably due to the interaction between catechin and milk components, or other factors such as degradation of catechin.15,16 Such interaction with milk proteins, specifically caseins, has been reported by other researchers17–20 and is considered as one major reason for loss of phenolic antioxidant activity, as well as degradation of green tea catechins under storage and digestive conditions.14,15 This can be attributed to catechins not being available to react with the reagents of the assays employed for measuring phenolic content and antioxidant activity, when association between catechins and milk proteins takes place. Whether such associations are reversible and affect the release of green tea antioxidants in the digestive tract remains debatable. In addition to the associations between milk proteins and green tea catechins, the association between milk fat and green tea catechins was lately demonstrated for the first time by Rashidinejad et al.21

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Recently, Rashidinejad et al.22 investigated the effect of free green tea extract on the pH, composition, phenolic properties, and antioxidant activity of a full-fat cheese. The retention of the individual catechins in the cheese curd, as well as recovery from the cheese digesta (digested in a simulated gastrointestinal digestion system), were examined. The results showed that although green tea extract (250–1000 ppm) in free form was able to increase phenolic content and antioxidant activity of the full-fat hard cheese, the increase was not proportional to the increasing concentration of green tea extract, probably due to the interactions between green tea catechins and milk components. The electron micrographs and Fourier transform infrared spectroscopy (FTIR) spectra of the cheese samples also provided evidence for hydrophobic association between milk fat and green tea catechins. The recovery of different catechins was also measured, and epigallocatechin gallate (EGCG), as the most potent green tea antioxidant, was not recovered from the cheese digesta. The incorporation of green tea catechins in free form may also affect the bioavailability of some of the nutrients in foods such as dairy products, and thus the nutritional value of the food may be compromised.12,19,20 Giroux et al.12 reported that the texture and sensory properties of Cheddar-type cheese, such as colour, hardness, and flavour, were significantly affected in a dose-dependent manner over a 29-day storage period by the addition of free (unencapsulated) green tea extract. Neither the interactions between green tea catechins and milk components, nor the recovery of different catechins in green tea extract, were measured. These researchers also found that green tea extract increased the astringency of the cheese during this period. Encapsulation of green tea catechins can be employed to minimise negative textural and flavour effects, and to increase retention within the cheese matrix.23 The encapsulation of bioactive compounds is recognised as a method to increase bioactivity, bioaccessibility, bioavailability, and stability.24 This is an industry-relevant approach to minimise the astringency of incorporated bioactive compounds in functional foods.25 We recently introduced a simple, fast, and inexpensive liposomal encapsulation method using soy lecithin to encapsulate green tea catechin and EGCG within low-fat hard cheese.23 This technology can help to minimise loss of antioxidant activity and prevent catechins from interacting with cheese components. The results demonstrated high values of encapsulation efficiency and encapsulation yields with good stability and retention of the encapsulated catechins. The current study determines the impact of the encapsulation process on the proximate composition and pH of full-fat cheeses fortified with liposomal encapsulated catechin (as the basic structure of all of the tea catechins) and green tea extract over a 90-day ripening period. The effect of liposomal encapsulation on the antioxidant activity and recovery of green tea catechins from digesta after in vitro simulated gastrointestinal digestion were measured to determine if there is efficacy in using this method to deliver antioxidant compounds in a cheese matrix.

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2. Materials and methods 2.1.

Milk, reagents, and chemicals

Pasteurized full-fat milk (3.3% fat) for cheese production was purchased from a local supermarket (Dunedin, New Zealand). Rennet was obtained from Renco (Eltham, New Zealand). Freeze dried mesophilic Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris starter culture (R-704) was obtained from Chr. Hansen (Hørsholm, Denmark). (+)-Catechin, (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), Trolox, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and gallic acid monohydrate were purchased from Sigma Aldrich (Auckland, New Zealand). (−)-Epigallocatechin gallate (EGCG) was supplied by Sapphire BioScience (Auckland, New Zealand). Green tea extract was obtained from Invita (Auckland, New Zealand). Soy lecithin was provided by Hawkins Watts (Auckland, New Zealand). Folin–Ciocalteu’s phenol reagent was obtained from Merck (Darmstadt, Germany). Fluorescein and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) were obtained from Eastman Kodak (Kingsport, TN, USA) and Cayman (Ann Arbor, MI, USA), respectively. Methanol (HPLC grade) was from Thermo Fisher Scientific (Auckland, New Zealand). All other chemicals used were of analytical reagent grade. 2.2.

Preparation of encapsulated antioxidants in liposomes

Aqueous solutions of catechin and green tea extract were prepared in 0.25 M acetate buffer ( pH 3.8) and coated with soy lecithin following the method of Rashidinejad et al.23 Briefly, soy lecithin (1%, w/v) was dispersed in the acetate buffer containing catechins with magnetic stirring (30 min) and then homogenized using a high shear blender (Polytron, PT-MR 2100, Kinematica, Switzerland) at 24 000 rpm (5 × 1 min bursts). Two controls, including catechin solutions and soy lecithin, and one blank (only acetate buffer) were set up for comparison. The prepared liposomes were stored at 4 °C for 24 h before being added to milk or used for further characterization. 2.3.

Experimental design for cheesemaking

The experiment consisted of five treatments (each repeated four times) including, (1) Con: control full-fat cheese made from full-fat milk without added catechins but with empty liposomes, (2) 250 ppm (+)-catechin cheese: full-fat cheese containing 250 ppm catechin encapsulated in liposomes, (3) 500 ppm (+)-catechin cheese: full-fat cheese containing 500 ppm catechin encapsulated in liposomes, (4) 500 ppm green tea extract cheese: full-fat cheese containing 500 ppm green tea extract encapsulated in liposomes, (5) 1000 ppm green tea extract cheese: full-fat cheese containing 1000 ppm green tea extract encapsulated in liposomes. There were 20 vats of cheeses randomized in five different cheesemaking sessions. 2.4.

Cheese manufacture

Full-fat cheeses were made with pasteurized full-fat milk, mesophilic culture, and rennet in a GR150 water bath (Grant, Cambridge, England) set at 31 °C, then packaged, ripened, and divided into three parts and vacuum packed separately in


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foil pouches (Audio Vac, Weesp, The Netherlands) as previously reported.26 Milk was stirred at 500 rpm in the vats using digital overhead stirrers while the pH of milk in each vat was monitored and recorded every 5 min. The cheeses were stored in a cool chamber at 8 ± 2 °C for 90 days of ripening. Samples were subsequently taken from each cheese on days 0, 30, and 90 and stored at −80 °C until further analyses. 2.5.

The pH and composition of the manufactured cheese

The pH values of milk and whey were measured on the day of cheese manufacture. Cheese samples (4–5 g) on days 0, 30, and 90 were ground in a mortar and pestle with a 1 : 1 aliquot of water to prepare a slurry, and the pH probe was inserted to directly measure pH. The moisture, fat, and protein contents of cheeses were measured in triplicate on day 0.27 Cheese yield was calculated from eqn (1): Cheese yield ð%Þ ¼

Weight of manufactured curd 100 Weight of initial milk

ð1Þ

2.6. Digestion of cheese samples in a simulated gastrointestinal digestion model An in vitro gastrointestinal digestion system, composed of gastric ( pH 1.2) and intestinal ( pH 6.8) digestion, was used to digest the cheese samples to mimic the role of the human digestive tract.13 The digestion process took place over 6 h at the constant temperature of 37 °C for both gastric and intestinal digestion. At the end of the digestion process, the digesta of each cheese sample was collected, filtered, and used for further analysis. Cheese samples were digested in triplicate.

coloured ferrous TPTZ. Acetate buffer (300 mM; pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl3·6H2O solution in deionized water were used as the reagents. The assay was carried out at 37 °C by adding diluted filtered (0.45 µM) samples (6.5 µL) and pre-heated (37 °C) FRAP reagent (193.5 µL) to each well of the microplate. The absorbance was read at 593 nm over 2 h. For ORAC antioxidant activity, a 0.02 M stock solution of Trolox was used as a control standard and appropriate working solutions were made as detailed in Rashidinejad et al.26 Fluorescein and AAPH working solutions were prepared from the corresponding stock solutions in 75 mM phosphate buffer ( pH 7.4). The diluted samples were added to the 96-well microplate with the fluorescein working solution, incubated for 30 minutes, and AAPH reagent was added. The microplate was transferred into the plate reader (37 °C) for recording the fluorescein intensity over 2 h (1 minute intervals) at 485 nm excitation and 527 nm emission. TPC, FRAP, and ORAC values were expressed as mg gallic acid equivalents per 100 g, mmol FeSO4 equivalent per kg, and micromoles Trolox equivalents (Teq) per g of fresh cheese samples, respectively.

2.8.

Retention coefficient of added phenolics

The retention coefficients of five major catechins, including (+)-catechin, EGCG, EGC, ECG, and EC, in green tea extract were calculated by subtracting the residual of the corresponding catechin detected in whey using high-performance liquid chromatography (HPLC), from the total catechin concentration initially added to milk, using eqn (2):

Catechin concentration in curd Initial catechin concentration in milk ðInitial catechin concentration in milkÞ ðCatechin concentration detected in wheyÞ ¼ Initial catechin concentration in milk

Retention coefficient of phenolic compound in curd ¼

2.7. Determination of total phenolic content and antioxidant activity Total phenolic content (TPC) and antioxidant activity of the digesta for all cheese samples were measured using the ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays13 in a 96-well microplate reader (KC4 Multi-Mode, BioTek, Winooski, VT, USA). For TPC, diluted digested cheese samples (20 µL) and standard gallic acid solutions were transferred into each well of the micro plate and reacted with Folin–Ciocalteu phenol reagent (100 µL; 10× diluted) in each well for 5 min at 25 °C. After that, 80 µL of 7% (w/v) Na2CO3 solution was added and the plate was immediately transferred into the plate reader (BioTek) to read the absorbance at 750 nm for 35 min (1 min intervals). FRAP antioxidant activity was carried out based on the reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to blue-


ð2Þ

The concentration of different catechins from green tea was determined by HPLC on a system equipped with a diode array detector (Agilent Technologies 1200 Series, Diegem, Belgium) following the isocratic method of Li et al.28 The mobile phase consisted of 0.1% trifluoroacetic acid in deionized water ( pH 2.0) and methanol at a volume ratio of 75 : 25 with a flow rate of 0.8 mL min−1 using an injection volume of 20 mL. Different concentrations (31–500 ppm) of catechin, EC, ECG, EGC, and EGCG were used as the standards. Chromatographic peaks of analytes were determined at 280 nm and identified by comparing the retention times with those of the external standards of each catechin, where peak integration using the external standard method was employed for quantification. 2.9.

In vitro recovery

The identification and quantification of catechin, EGCG, EGC, ECG, and EC in the digested cheese samples were carried out

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using the HPLC method as described in the last section, and the corresponding recoveries were calculated using eqn (3):


Recovery of catechin ð%Þ ¼

Catechin recovered from digesta of corresponding cheese sample Initial catechin concentration in corresponding cheese Catechin recovered from digesta of corresponding cheese sample 100 ðInitial catechin concentration in milk retention coefficientÞ cheese yield

2.10. Transmission electron microscopy Cheese samples were sliced into cubes (1–2 mm) and placed into baskets of a Lynx EL tissue processor (Australian Biomedical Corporation Ltd, Mount Waverley, Victoria, Australia). Approximately three pieces for each specimen were suspended in a small, sealed container containing 2 mL of 4% aqueous osmium tetroxide and held for 17 days at 4 °C, allowing no contact of the liquid osmium tetroxide with the specimens. These were then vapour-fixed and removed from the baskets. Afterwards, the specimens were embedded in 3% agarose and placed into new baskets. The specimens were then treated in the Lynx EL tissue processor by further fixing using 3% glutaraldehyde and 3% paraformaldehyde in 0.1 M piperazine-N,N′bis(2-ethanesulfonic acid) buffer for 2 h, then washed in the buffer and kept in 1% osmium tetroxide for 1 h. The specimens were then washed with water and stained in 0.5% aqueous uranyl acetate for 10 h before washing again in water and dehydrating through a graded ethanol series up to 100% ethanol. The specimens were embedded in Spurr’s resin (approximately 40 h) and were then removed from the tissue processor and cured in solid resin blocks (60 °C for 48 h). Sections of 80 nm thickness from the blocks were cut by use of a Leica UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and mounted on a 300 mesh copper grid before contrasting with uranyl acetate and lead citrate using an LKB 2168 Ultrostain grid stainer (LKB-Produkter AB, Bromma, Sweden). Lastly, the sections were viewed using a Philips CM100 transmission electron microscope (TEM; Philips Electron Optics, Eindhoven, The Netherlands) at an accelerating voltage of 100 kV. A MegaView 3 digital camera (Soft Imaging System GmBH, Münster, Germany) was used to take the micrographs.

2.11. Fourier transform infrared spectroscopy Control cheese, and cheeses containing 500 ppm liposomal encapsulated catechin and green tea extract were examined by Fourier transform infrared (FTIR) spectroscopy analysis according to the method of Chen and Irudayaraj.29 This technique was employed to determine interactions between green tea catechins and cheese fat (which could occur by leakage of catechins from the liposomes) by comparing spectra for treated cheese samples with the control. Frozen cheese samples were thawed and placed on the surface of a diamond chloride crystal in the light path of a purged Mattson Polaris™ FTIR spectrometer (Varian 3100 FT-IR Excalibur series, Palo Alto, CA, USA). A slurry of aluminium oxide in water ( particle

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size 0.05). The results are in agreement with the effect of free green tea catechins on the composition of cheese.26 Likewise, the pH of whey from different cheeses with different treatments remained unchanged (Table 2) in contrast with the pH results presented previously,26 where the free form of catechin incorporated into low-fat cheese decreased the pH of cheese during both the manufacturing process and ripening period (90 days). Hence, catechin and green tea extract did not affect either cheese manufacture or ripening, and therefore likely remain entrapped within the nanoliposomes. The observations in the current experiment contrast with those reported by Giroux et al.12 and Gad and El-Salam;30 however, in both of these studies, the catechins were not encapsulated. Giroux et al.12 demonstrated that the addition of 1–2 g kg−1 free green tea extract decreased the pH of a Cheddar-type cheese. Han et al.10


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Effect of liposomal encapsulated (+)-catechin and green tea extract on the composition, weight, and yield of full-fat hard cheesea

Composition Moisture (%) Protein (%) Fat (%) Cheese weight (g) Whey weight (g) Cheese yield (%)


Paper

Con

250 ppm C a

62.3 ± 0.7 14.2 ± 0.8a 19.2 ± 1.0a 92.0 ± 1.6a 388 ± 3a 18.41 ± 0.21a

500 ppm C

a

a

63.5 ± 0.6 14.3 ± 1.3a 18.3 ± 0.5a 92.9 ± 1.5a 386.4 ± 2.3a 18.57 ± 0.11a

62.1 ± 0.4 15.2 ± 0.7a 19.07 ± 0.28a 90.9 ± 1.2a 388 ± 4a 18.19 ± 0.18a

500 ppm GTE

1000 ppm GTE

a

62.7 ± 0.8a 14.8 ± 1.4a 18.4 ± 0.5a 92.1 ± 0.6a 385.7 ± 2.1a 18.42 ± 0.13a

62.73 ± 0.28 15.6 ± 0.9a 18.1 ± 0.6a 91.9 ± 1.5a 387 ± 5a 18.38 ± 0.09a

a Means of four vats with three replicates of compositional measurements. Con: cheese made of full-fat milk without added (+)-catechin (C) or green tea extract (GTE), 250 ppm C: cheese made of full-fat milk with 250 ppm added encapsulated (+)-catechin, 500 ppm C: cheese made of fullfat milk with 500 ppm added encapsulated (+)-catechin, 500 ppm GTE: cheese made of full-fat milk with 500 ppm added encapsulated GTE, 1000 ppm GTE: cheese made of full-fat milk with 1000 ppm added encapsulated GTE. Means within a row with the same superscript letters are not significantly different (P > 0.05).

Table 2 Effect of liposomal encapsulated (+)-catechin and green tea extract on the pH of full-fat hard cheese during the manufacturing process and ripening perioda

Con Time 0 (milk pH at 31 °C) Immediately after addition of buffer containing catechin 10 min after adding culture 20 min after adding culture 30 min after adding culture Immediately after cutting curd 10 min after cutting curd 20 min after cutting curd 30 min after cutting curd Whey (after drainage) Cheese (day 0) Cheese (day 30) Cheese (day 90)

250 ppm C a

6.62 ± 0.01 6.54 ± 0.01a 6.49 ± 0.02a 6.41 ± 0.02a 6.37 ± 0.02a 6.00 ± 0.01a 5.98 ± 0.03a 5.86 ± 0.04a 5.75 ± 0.03a 5.50 ± 0.04a 5.40 ± 0.01a 4.81 ± 0.02a 4.98 ± 0.03a

a

6.62 ± 0.02 6.52 ± 0.03a 6.48 ± 0.01a 6.42 ± 0.01a 6.38 ± 0.01a 5.98 ± 0.02a 5.96 ± 0.02a 5.85 ± 0.03a 5.73 ± 0.02a 5.52 ± 0.02a 5.39 ± 0.03a 4.79 ± 0.01a 4.97 ± 0.05a

500 ppm C a

6.63 ± 0.01 6.53 ± 0.01a 6.47 ± 0.03a 6.42 ± 0.01a 6.37 ± 0.01a 6.01 ± 0.01a 5.95 ± 0.05a 5.84 ± 0.02a 5.74 ± 0.04a 5.49 ± 0.03a 5.41 ± 0.02a 4.81 ± 0.03a 5.00 ± 0.07a

500 ppm GTE a

6.63 ± 0.01 6.54 ± 0.01a 6.51 ± 0.02a 6.45 ± 0.01a 6.39 ± 0.01a 6.04 ± 0.01a 6.01 ± 0.02a 5.87 ± 0.03a 5.73 ± 0.02a 5.54 ± 0.03a 5.42 ± 0.01a 4.80 ± 0.02a 5.01 ± 0.05a

1000 ppm GTE 6.62 ± 0.02a 6.52 ± 0.03a 6.48 ± 0.02a 6.44 ± 0.02a 6.37 ± 0.01a 6.02 ± 0.02a 6.00 ± 0.02a 5.89 ± 0.02a 5.71 ± 0.03a 5.53 ± 0.01a 5.44 ± 0.02a 4.82 ± 0.03a 4.97 ± 0.05a

a Means of four vats with three replicates of compositional measurements. Con: cheese made of full-fat milk without added (+)-catechin (C) or green tea extract (GTE), 250 ppm C: cheese made of full-fat milk with 250 ppm added encapsulated C, 500 ppm C: cheese made of full-fat milk with 500 ppm added encapsulated C, 500 ppm GTE: cheese made of full-fat milk with 500 ppm added encapsulated GTE, 1000 ppm GTE: cheese made of full-fat milk with 1000 ppm added encapsulated GTE. Means within a row with the same superscript letters are not significantly different (P > 0.05).

also reported that the addition of unencapsulated catechins, including catechin, could decrease the pH of the rennet gels made from commercial pasteurized milk (3.25% fat and 3.2% protein, w/v). Cheese pH is one of the important factors dictating texture and flavour because of the impact upon ripening reactions, bacterial growth, and protein aggregation.31–33 Changes in pH during cheese manufacture were expected in the control group.14 In the current study, the drop in pH during cheese manufacture was similar for all treatments and the control. Therefore, encapsulation of catechins can be used to protect phenolic compounds from interacting with milk and cheese components, and accordingly, minimise changes to cheese pH that would otherwise occur by the release of catechins into the cheese matrix.12 Rupture of nanoliposomes within cheese during the ripening period was therefore not considered to be significant, as there was no change to the pH of cheese. Nanoliposomes were therefore stable within milk and curd during the process of cheese manufacture, as well as within cheese during the subsequent ripening period.


3.2.

Total phenolic content and antioxidant activity

The results of TPC, FRAP, and ORAC for control and catechintreated cheeses following digestion are presented in Fig. 1. TPC and antioxidant activity increased in the control cheeses during storage, in agreement with Rashidinejad et al.26 These results also confirm that the nanoliposomes containing green tea catechins were ruptured during simulated digestion, releasing the catechins into the digesta, in agreement with the results of Taylor et al.34 The addition of both catechin and green tea extract significantly (P ≤ 0.05) increased the TPC and antioxidant activity (as measured by both FRAP and ORAC) of full-fat cheese over the 90-day ripening period in a proportional manner with concentration, compared with the diminished increase observed when free catechins were added,13,26 which was possibly due to free phenolic interactions with milk components, or instability during ripening. It should be noted that, although the incorporation of green tea catechins in free form may affect the antioxidant activity, and correspondingly, bioavail-

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Fig. 1 Total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) antioxidant activity of the control and catechin-treated full-fat cheeses over 90 days of ripening. TPC in units of gallic acid equivalents (GA eq.) per 100 g, FRAP in units of FeSO4 equivalent (mmol kg−1), and ORAC in units of Trolox equivalents per gram (Teq, µmol g−1) of cheese weight. Con: cheese made of full-fat milk without added catechin but with the same amount of empty liposomes; 250 ppm catechin: cheese containing 250 ppm (+)-catechin encapsulated in liposomes; 500 ppm catechin: cheese containing 500 ppm (+)-catechin encapsulated in liposomes; 500 ppm GTE: cheese containing 500 ppm green tea extract (GTE) encapsulated in liposomes; 1000 ppm GTE: cheese containing 1000 ppm GTE encapsulated in liposomes; columns for the same analysis assay (i.e. TPC, FRAP, or ORAC) with different letters are statistically different at the P ≤ 0.05 level. Error bars are standard deviations.

ability of green tea catechins,12,19,20 some researchers report that the associations between catechins and milk proteins can be considered as a positive effect by which catechins

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enhance the functionality and/or bioavailability of milk proteins.18 In the current study, the purpose was not to increase the bioavailability of cheese nutrients, but rather to enhance


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the bioavailability of green tea antioxidants by means of nanoliposomal encapsulation. As shown in Table 3, both FRAP and ORAC are highly correlated with TPC, as well as with each other. The correlation between FRAP or ORAC and TPC is not as strong as between FRAP and ORAC. This may be due to some endogenous compounds, such as peptides and amino acids, reacting with the Folin–Ciocalteu reagent in the TPC method, and thus considered as phenolic compounds, but without showing antioxidant activity in FRAP and ORAC tests. Fig. 1 also shows that cheese fortified with 500 ppm encapsulated catechin has similar TPC values to the cheese fortified with 1000 ppm green tea extract; catechin used in this experiment was a pure (>99%) analytical antioxidant compound whereas the green tea extract was a crude extract of green tea and will contain other compounds. In addition, and as described previously,13,26 cheeses fortified with 1000 ppm green tea extract showed a significantly (P ≤ 0.05) higher antioxidant activity (both FRAP and ORAC) than cheese fortified with 500 ppm pure catechin, even though they showed almost similar TPC values on day 90 of the ripening period, indicating that EGCG, EGC, and ECG in green tea extract have higher antioxidant activities than catechin. Frei and Higdon8 stated that EGC, EGCG, and ECG were better antioxidants than catechin in lipid systems.

3.3. HPLC analysis, retention coefficient, and recovery of the green tea antioxidants HPLC profiles of different concentrations (31–500 ppm) of five standard catechin derivatives (catechin, EC, ECG, EGC, and EGCG), and green tea extract are shown in Fig. 2. HPLC analysis showed that there was no trace of (+)-catechin or any of the catechins in green tea extract in the whey samples from the cheese made with encapsulated polyphenols; thus, it can be hypothesised that the liposomal encapsulated green tea catechins were completely retained in the full-fat cheese. This is in contrast to free catechins added to low-fat cheese, where only 70% was retained in the curd at 500 ppm fortification,26 and also in contrast to free green tea extract added to fullfat cheese.13 As the liposomes manufactured in this experiment were mainly composed of phospholipids, similar to those found in cheese fat, they may adsorb to the cheese fat

Table 3 The correlations between total phenolic content and antioxidant activity of full-fat hard cheeses fortified with encapsulated (+)-catechin or green tea extracta

Ripening period

TPC/FRAP

TPC/ORAC

FRAP/ORAC

Day 0 Day 30 Day 90

0.915 0.887 0.908

0.856 0.873 0.885

0.928 0.987 0.974

a TPC: total phenolic content, FRAP: ferric reducing antioxidant power, ORAC: oxygen radical absorbance capacity; n = 6, compilation of the cheese samples containing both (+)-catechin and green tea extract.


globules containing surface phospholipids via the polar headgroups and be retained in the curd rather than partitioning into the whey. In a report examining free green tea extract,22 free gallated catechins, including EGCG, EGC, and ECG, had very high retention coefficients (0.81 to 1) in full-fat cheese, whereas two non-gallated catechins in free form, catechin and EC, had retention coefficients of 0.43 to 0.69. This could occur because of different affinities of green tea catechins to milk components, as well as differing stability during cheesemaking. The recovery results are confirmed by both TPC and antioxidant activity results reported in section 3.2. Table 4 shows the recovery of encapsulated catechin, and the different catechins in green tea extract after simulated digestion. Pure catechin showed the highest recovery, with almost half being recovered when it was added at an initial level of 500 ppm. The different catechins from green tea were recovered in descending order of EC > catechin > ECG ≈ EGC > EGCG, in most cases (Table 4). This order of recovery for different green tea catechins is consistent with the results observed by Green et al.35 who studied the recovery and stability of different green tea catechins in a simulated gastric and intestinal digestive model, and reported that EGCG and EGC green tea catechins were the most susceptible catechins to degradation during in vitro digestive conditions. Galloylated tea catechins are known to be one of the least absorbed natural polyphenols in the human gut.36 The very low recovery of compounds, such as EGCG and EGC, could be due to hydrolysis and methylation occurring during digestion; e.g. EGCG can be hydrolysed to gallic acid and EGC, and EGC can be methylated to other compounds that might not be considered as catechins.37 Although the recovery values obtained in the present study for some compounds, such as EGCG, are low, these are higher than those found by Green et al.35 where the in vitro recovery of EGCG and EGC was