Food Hydrocolloids 81 (2018) 364e370

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Liposomal dispersion and powder systems for delivery of cocoa hull

waste phenolics via Ayran (drinking yoghurt): Comparative studies on
in-vitro bioaccessibility and antioxidant capacity
€
Gokce Altin a, Mine Gültekin-Ozgüven a
, Beraat Ozcelik a, b, *
a
Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Maslak, 34469, Istanbul, Turkey
b
BIOACTIVE Research & Innovation Food Manufac. Indust. Trade Ltd., Katar Street, Teknokent ARI-3, B110, Sarıyer, 34467, Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Cocoa hull waste phenolic extract (CHWPE) was encapsulated by chitosan coated liposomes with 0.2% of
Available online 28 February 2018 concentration. Ayran (drinking yoghurt) system was employed as a vehicle for inclusion of phenolic
loaded liposomes in dispersion and powder form. Antioxidant capacityand in-vitro bioaccessibility of
Keywords: both dispersion and spray-dried powder liposomal systems with CHWPE included in ayran samples were
Phenolics investigated during the storage period of 15 days. Compared to the ayran sample with CHWPE (0.05%),
Liposomal encapsulation
in-vitro bioaccessibility of encapsulated CHWPE in ayran was determined to be 5 fold and 2 fold higher in
Ayran(drinking yoghurt)
the form of liposomal powder (0.05%), and liposomal dispersion (0.1%) in terms of total phenolics and
In-vitro bioaccessibility
Stability
total flavonoids, and total antioxidant capacity (DPPH and CUPRAC assays). The bioaccessibility of
catechin (50%) and ferulic acid (80%) of encapsulated CHWPE in liposomal powder showed no significant
difference (p > 0.05) during storage. In dispersion of liposomes, bioaccessibility of individual phenolic
compounds was variable. Vanillin showed the minimum bioaccessibility in liposomal powder (19%) and
liposomal dispersion (16%) at the end of storage. In summary, the use of liposomal delivery systems in
powder form should be recommended in ayran system. Although the psychochemical properties of
enriched ayran were not monitored in this study, it is suggested that these properties be taken into
consideration in determining the form of liposomes to be added to a food product.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction Sur & Panda, 2017). On the other hand, they are highly instable
bioactive compounds due to exposure to degradation by light,
Polyphenols are the secondary metabolites of plants including soluble oxygen or enzymes. In addition, they may interact with
cocoa. These compounds exhibit positive effects on human health. other food components such as proteins and carbohydrates, hence
Due to their antioxidant; antimicrobial, anti-inflammatory, anti- they may loose their activity. In this point, encapsulation technol-
osteoporotic and hepatoprotective activity, they act as a protective ogy is an effective way to inhibit the interaction between poly-
agent against some chronic diseases such as cancer, type-II dia- phenols and other molecules. Moreover, this technology protects
betes, cardiovascular disease or non-alcholic fatty liver diseases polyphenols from adverse environmental conditions and also
€
(Tezcan, Gültekin-Ozgüven, €
Diken, Ozçelik, B. & Erim, 2009; provides a mask of the odor and color of polyphenols (Fang &
Mahomoodally & Ramalingum, 2015; Wadhawan & Anand, 2016; Bhandari, 2010). As an encapsulation technique, liposomes can
allow successful delivery of phenolic compounds. Liposomes are
enclosed spherical vesicles formed by dispersion of certain polar
lipids in an aqueous solvent. Therefore, they can be organized by in
Abbreviations: CHWPE, cocoa hull waste phenolic extract; TPC, total phenolic
content; TAC, total antioxidant content; TFC, total flavonoid content; UHPLC, ultra-
one or several concentric phospholipidic bilayers with an internal
high pressure dispersion chromatography; PDA, photodiode array detector. aqua phase which makes them a carrier system for both water and
* Corresponding author. Istanbul Technical University, Faculty of Chemical and oil soluble functional compounds (Mozafari et al., 2008). Moreover,
Metallurgical Engineering, Department of Food Engineering, 34469, Maslak, liposomes are biodegradable, biocompatible and non-
Istanbul, Turkey.
immunogenic, so they have a great potential for applications in
E-mail addresses: [email protected] (G. Altin), [email protected]
€
(M. Gültekin-Ozgüven), [email protected] (B. Ozcelik). pharmaceutical, food and agricultural industries (McClements & Li,

https://doi.org/10.1016/j.foodhyd.2018.02.051
0268-005X/© 2018 Elsevier Ltd. All rights reserved.
 G. Altin et al. / Food Hydrocolloids 81 (2018) 364e370 365

2010). However, they consist of flexible and fragile bilayer mem- 24.3 ± 2 mg TEAC/g, 12.41 ± 3 mg TEAC/g in terms of CUPRAC and
branes (Jeon, Young, & Park, 2015). To overcome this challenge, DPPH assays, respectively. To obtain uncoated liposome dispersion
coating the liposome surface with a polymer material is an effective with CHWPE, 2% (w/v) of lecithin in acetate buffer (pH ¼ 3.5 ± 0.1;
solution to ensure the protection of their stability. Layer-by-layer 0.1 mol/L) was stirred overnight at room temperature. Based on our
deposition method is one of the coating methods which based on previous study (Altin et al., 2018), 0.2% (w/v) concentration of
interaction between the opposite surface charges (Ciobanu et al., CHWPE with 73.6% encapsulation efficiency was selected for this
2007). Chitosan, pectin or combined chitosan/pectin coatings can study. CHWPE (0.2% w/v) was added to this lecithin solution and
be used as protective coatings in the acidic milieu of the stomach dissolved. Uncoated liposome dispersions with CHWPE and
and triggered release systems in the colon (Gibis, Jeen, & Weiss, without CHWPE (blank) were prepared by a high shear disperser
2014). Still, it is not easy to use such liquid systems in food for- (DI-25 Yellowline, IKA) for 10 min at 9.500 rpm followed by passing
mulations. We showed that when liposome encapsulated phenolic five times through a high pressure homogenizer (Microfluidizer
compounds were dried, they became more feasible to be used in Processor M-110L, Microfluidics, Newton, USA) at homogenization
€
food formulation successfully (Gültekin-Ozgüven, Karadag, Duman, pressure of 25.000 psi. Layer-by-layer method was applied to coat
Ozkal, & Ozcelik, 2016). Drying of liposomal systems enhances liposomes with chitosan. Uncoated liposomes with CHWPE and
physico-chemical stability and storage (Moraes et al., 2012). without CHWPE (blank) were added to chitosan solution (0.8%, w/
Moreover, drying process further increased in-vitro bioaccessibility v) dissolved in acetate buffer (pH ¼ 3.5 ± 0.1; 0.1 mol/L) with a ratio
of the phenolics beyond that involved in the encapsulation process of 1:1 (w/w) and stirred overnight at room temperature. By this
€
(Altin, Gültekin-Ozgüven, & Ozcelik, 2018). way, negatively charged liposomes were coated with positively
Ayran (drinking yoghurt) is one of the traditional yoghurt €
charged chitosan layer (Gültekin-Ozgüven et al., 2016).
products which is prepared by addition of water to yoghurt or
addition of yoghurt culture (Streptococcus thermophilus and 2.3. Preparation of liposomal powder systems with and without
L. delbrueckii subsp. Bulgaricus) to standardized milk according to CHWPE
the Turkish Food Codex (2009). In the current study, ayran system
was employed as a vehicle for inclusion of liposomes in dispersion Only coated liposome dispersions with and without CHWPE
and powder form with phenolics to increase antioxidant capacity were spray dried since addition of MD to uncoated ones immedi-
and enhance in-vitro bioaccessibility of phenolics in food formu- ately caused extensive flocculation and a complete breakdown of
lation. Since phenolic profile of plain yoghurt was very different €
the system (Altin et al., 2018; Gültekin-Ozgüven et al., 2016). Prior
from cocoa hull phenolics, it was an effective food matrix to to spray drying process, coated liposome dispersions with CHWPE
monitor changes in in-vitro bioaccessibility and antioxidant ca- 0.1% (w/v) and without CHWPE were mixed with MD solution (40%
pacity of phenolics during storage. In this concept, encapsulated w/v in acetate buffer) with a ratio of 1:1 (w/w). Feeding solution
cocoa hull waste phenolic extract (CHWPE) characterized in our contained 20% (w/v) MD, 0.5% (w/v) lecithin, 0.05% (w/v) CHWPE
previous study was employed (Altin et al., 2018). Stability and in- and 0.2% (w/v) chitosan for spray drying process. CHWPE powder
vitro bioaccessibility of the encapsulated CHWPE in ayran samples was also prepared as a control sample. For this aim, freeze dried
was observed during storage of 15 days at þ4  C. To our knowledge, CHWPE 0.05% (w/v) was spray dried after mixing with MD (20 g/
this is the first study on comparison of both liposomal dispersion 100 g). Based on our previous studies (Altin et al., 2018; Gültekin-
and liposomal powder applied in a food system. €
Ozgüven et al., 2016; Karadag et al., 2013), a laboratory scale
spray dryer (Mini spray dryer B-290, BUCHI, Switzerland) was used
2. Materials and methods for drying process. It equipped with a 1.5-mm nozzle atomizer
operated at an atomizing air flow of 5 cm3/min, a feed rate of
2.1. Materials 2.5 cm3/min at an inlet temperature of 170  C resulting in an outlet
temperature of 75e80  C, and 0.67 m3/min air flow. Obtained
Cocoa hull waste was provided by a local chocolate factory in powder samples were stored in airtight containers and placed in a
Turkey. After roasting of cocoa beans, cocoa hull waste was trans- desiccator at room temperature. Reported in our previous study
ported to the laboratory under vacuum package. Ayran was pur- (Altin et al., 2018), the amount of CHWPE was 110.85 ± 28.36 mg/L
chased from a local supermarket. Lecithin (Soybean phospholipids, interior and 24.98 ± 7.3 mg/L on surface of spray-dried liposomal
97%- Ultralec® P) was provided by Rotel, Turkey. Chitosan with 80% powders, respectively.
DDA (degree of deacylation) was donated from Primex (Siglufjor-
dur, Iceland). Low molecular weight maltodextrin (MD), with a 2.4. Measurements of particle size distribution and zeta (z)
dextrose equivalent value of 20, was a gift from Tunckaya Kimyevi potential
Maddeler Ticaret ve Sanayi Inc., Turkey. Sephadex G50 was sup-
plied from GE Healthcare Life Sciences (Uppsala, Sweeden). Gallic Liposome suspensions were sized by a static light scattering
acid, neocuproine, trolox, 2,2-diphenyl-1-picrylhydrazy reagent, instrument (Mastersizer 2000, Malvern Instruments). Average
copper (II) chloride, catechin were purchased from Sigma-Aldrich particle diameters was reported by using the volume mean diam-
Co. (St. Louis, USA). Folin Ciocalteu’s phenol reagent was supplied eter (d4,3). The surface charge of the particles was determined using
from Merck KGaA (Darmstadt, Germany). Triton X100 was pur- a Zetasizer® 2000 (Malvern Instruments Ltd.,Worcestershire, UK).
chased from Carl Roth GmbH (Karlsruhe, Germany). SPE cartridges Liposomal powders with and without CHWPE and CHWPE powder
were obtained from Macherey-Nagel (Duren, Germany). were reconstructed in acetate buffer (pH ¼ 3.5 ± 0.1; 0.1 mol/L)
with a ratio of 1:5 (w/v) before the measurements.
2.2. Preparation of dispersion liposomal systems with CHWPE
2.5. Inclusion of liposomal systems into ayran formulation
CHWPE was obtained by methanol/Milli-Q water (80%, v/v)
according to Azizah, Nik Ruslawati, and Swee Tee (1999) and freeze Ayran samples containing freeze-dried CHWPE (S1) (0.05%),
dried (Christ Alphna 1-2 LDplus, Osterode am Harz, Germany). spray dried CHWPE powder (S2) (0.05%), chitosan coated liposomal
Freeze-dried CHWPE contained 22.6 ± 2 mg GAE/g of phenolics, dispersion with CHWPE (S3) (0.1%), and spray dried liposomal
5 ± 0.1 mg CE/g of flavonoids and exhibited antioxidant capacity of powder with CHWPE (S4) (0.05%) were prepared freeze-dried and
366 G. Altin et al. / Food Hydrocolloids 81 (2018) 364e370

encapsulated liposomal systems were added into ayran with a ratio 2.8.3. Determination of total antioxidant capacity (TAC)
of 1:10 (freeze-dried and encapsulated liposomal systems with Cupric ion reducing antioxidant capacity (CUPRAC) assay (Apak,
CHWPE/ayran) (w/v). To eliminate the possible interferences dur- Guclu, Ozyurek, & Karademir, 2004) and 2,2-diphenyl-1-picrylhy-
ing experiments, blank samples (liposome dispersions without drazyl (DPPH) assay (Kumaran & Karunakaran, 2006) were applied
CHWPE and liposomal powders without CHWPE) were also added to determine TAC. In CUPRAC assay, 100 mL of sample was treated
to ayran with the same ratio (1:10 w/v). The stability and in-vitro with 1 mL of ammonium acetate (pH:7), 1 mL of neocuproine so-
bioaccessibility of ayran samples with both dispersion and powder lution (7.5  103 mol/L), 1 mL of copper (II) chloride solution
liposomal systems were observed during the storage period of (102 mmol/L) and 1 mL of distilated water, respectively. The
ayran at 4  C in the dark. Samples were collected 5 days of intervals mixture was left to stand for 25 min in dark and the absorbance was
(1st, 5th and 10th and 15th day). measured at 450 nm. In DPPH assay, 2 mL of DPPH solution
(101 mmol/L) were added to 100 mL of sample and shaken for 10 s.
The mixture was left to stand for 25 min in dark and absorbance
2.6. Extraction of phenolics from ayran samples
was measured at 517 nm. All analyses were performed in triplicates
and the results were expressed as mg TEAC per L sample for both
Ayran samples (20 g) collected in the days of 1st, 5th and 10th
assays.
and 15th were mixed with 30 ml of acetone-HCL solution (30% (v/v)
acetone containing HCL 0.1% (v/v)), and kept at þ 4  C overnight.
2.9. Identification and quantification of CHWPE in ayran samples
After filtration through Whatman No:2 filter paper, the aqueous
during storage
phase was collected and aceton was removed by rotary evaporator
at 40  C (Karaaslan, Ozden, Vardin, & Turkoglu, 2011). The
Change in phenolic profile of both undigested and digested
remained extracts were centrifuged at 10.000 rpm for 2 min before
ayran samples were determined using an Ultra high pressure
freeze drying. Freeze dried extracts were stored at 80  C for
dispersion chromatography (UHPLC) (Shimadzu, CTO-10ASVP, 264
further analyses.
Kyoto, Japan) equipped with a SPD-M10A photodiode array detec-
tor (PDA) during storage. Reversed-phase chromatography was
2.7. In-vitro digestion performed with 250  4.6 mm Kromasil 100 C-18 column packed
with 5 mm particles (Teknokroma, Barcelona, Spain), fitted with a
In-vitro bioaccessibility studies were conducted according to the security guard C18 ODS (4  3.0 mm i.d). A gradient of mobile phase
method of Tan et al. (2014). Gastrointestinal condition was simu- A (MQ water containing %0.1 formic acid) and mobile phase B
lated in a shaking water bath (New Brunswick Scientific Co., Inc., (MeCN) was used for chromatographic separation (Altin et al.,
New Jersey, USA) at 37  C, 100 rpm. First, 1.5 mL of sample was 2018). The linear gradient was used as follows: 0e2 min: 95% B,
treated with 13.5 mL of basal saline containing 140 mmol/L NaCl 2e25 min: 5% B. The flow rate was 0.5 mL min1 and the operating
and 5 mmol/L KCl for 10 min. To initiate the gastric digestion, pH temperature was 40  C. The injection volume was 10 mL. The chro-
was adjusted to 2.0 using 1.0 mol/L NaOH before addition of 4.5 mL matogram was recorded at 286 nm. Before injection into UHPLC,
of simulated gastric fluids (3.2 g/L pepsin in 1 mol/L HCI, pH ¼ 2.0). the undigested samples were mixed with formic acid/methanol
Then, samples were incubated for 1 h at 37  C. To simulate intesti- (1:100, v/v) solution (1:1, w/v) and centrifuged at 10.000 rpm for
nal digestion process, pH was adjusted to 7.5 with 1.0 mol/L NaOH 2 min. After that, samples were filtered through 0.45 mL membrane
and the mixture treated with 4.5 mL simulated intestinal fluid filters. Before the analysis of digested samples, solid phase extrac-
containing 4.76 mg/mL pancreatin and 5.16 mg/mL porcine bile tion method was employed to purify them. For this aim, 500 mg/
extract in phosphate buffer solution for 2 h. Finally, the digested 6 mL C18 SPE cartridges (MACHEREY-NAGEL GmbH & Co.KG, Ger-
samples were centrifuged at 6000 rpm for 15 min. Top phase was many) were conditioned by addition of 6 mL of formic acid/meth-
collected and storaged at 80  C for further analyses. anol (1:100, v/v) and 4 mL of formic acid/MQ water (1:100 v/v),
respectively. Then, 1.5 mL of sample was acidified with 30 mL of
formic acid and then centrifuged at 12.000 rpm for 10 min before
2.8. Spectrophotometric assays
passing through the activated cartridges. Then, the cartridges were
exposed to 5 mL of formic acid/water (1:100, v/v). Remained
2.8.1. Determination of total phenolic content (TPC)
mixture in the cartridges was eluted with 1.5 mL of formic acid/
TPC was measured according to FolineCiocalteu reagent test
methanol (1:100, v/v) and filtered through 0.45 mL membrane fil-
described by Gibis, Vogt, and Weiss (2012). 200 mL of the diluted
ters before injection into UHPLC. All analyses were performed in
sample was mixed with 1.5 mL of the diluted FolineCiocalteau re-
triplicates and the results were expressed as mg/100 g.
agent (1:10 v/v). After addition of 1.2 mL of the sodium carbonate
solution (7.5 g/100 mL), the mixture was left to stand for 48 min in
2.10. Statistical analysis
dark. Absorbance was measured at 765 nm by a spectrophotometer
(BioTek instruments, Winooski, USA). All analyses were performed
IBM SPSS Statistics 24.0 software was employed for statistical
in triplicates and the results were expressed as mg gallic acid per L
analysis. All measurements were repeated at least three times using
sample.
triplicate samples. Differences were analyzed by Tukey’s Test
comparisons and p value of <0.05 was chosen to determine sig-
2.8.2. Determination of total flavonoid content (TFC) nificant differences.
250 mL of diluted sample was treated with 75 mL of sodium ni-
trite solution (5 g/100 mL) for 6 min. Then, 150 mL of aluminium 3. Results and discussion
chloride solution (10 g/100 mL) was added. At the 11th minute,
500 mL of sodium hydoxide (1 mol/L) and 2.5 mL of water were 3.1. Stability of encapsulated CHWPE in ayran during storage
added and the mixture was shaken for 10 s. Absorbance was
measured at 510 nm. All analyses were performed in triplicates. The Coated liposome dispersion with CHWPE with z epotential
results were expressed as mg catechins per L sample (Dewanto, of þ35.7 ± 0.92 mV at 300 nm particle size, and liposomal powder
Wu, Adamm, & Liu, 2002). with CHWPE with z epotential of þ34.9 ± 0.42 at 231 nm obtained
 G. Altin et al. / Food Hydrocolloids 81 (2018) 364e370 367

in our previous study was prepared (Altin et al., 2018). After in- (Trigueros, Wojdyło, & Sendra, 2014). pH of ayran was approxi-
clusion in ayran, changes in TPC, TFC and TAC values of CHWPE in mately 4.5 during storage. Regarding conservation of CHWPE in
ayran samples were observed in the 1st, 5th and 10th and 15th days each liposomal system during storage period, a significant decrease
(Table 1). Results showed that liposomal encapsulation technique (p < 0.05) was determined in TPC, TFC and TAC values in S1 and S2
showed a protective effect against degradation of CHWPE in ayran between the 1st and 15th days where CHWPE addition was per-
formulation during storage. Particularly, TPC, TAC and TFC values in formed in non-encapsulated form. In terms of TPC, CHWPE in S4
S4 were significantly different (p < 0.05) from other samples was stabile during storage. Meanwhile; liposomal powder provided
collected in the 1st, 5th, 10th and 15th days. By the way, TPC and TAC stability of CHWPE in ayran until the 10th day according to
TFC values of CHWPE in ayran samples were increased approxi- DPPH assay. In contrast, antioxidant capacity of CHWPE in terms of
mately 5 times by inclusion via liposomal powder during storage. CUPRAC assay decreased significantly at the 5th day in S4. Similar
Indeed, addition of CHWPE powder and liposomal dispersion into trend was observed for also TFC in S4, but this value did not change
ayran caused an increment in TPC and TFC at least 3 fold for both after 5 days up to the end of storage. On the other hand, TFC of
assays up to the end of storage period, but less than CHWPE in CHWPE in S3 did not change (p > 0.05) while a significant decrease
liposomal powder. Conversely, S1 sample did not show an increase (p < 0.05) was determined in terms of TPC during storage period,
in TFC. On the other hand, TPC value was 2 times higher in S1 than which might be related to the destruction of liposome structure in
control sample collected in the 1st and 5th day. Nevertheless, there dispersion. Previously, zeta potential of the acidified milkproteins
was no significant difference (p > 0.05) between S1 and control was reported to be in the range of 0 and -30 mV when pH was
sample in terms of TPC at the 15th day. Similarly, TAC of ayran was between 4.4 and 4.8 by Sejersena et al., 2007. The negative carboxyl
increased approximately 2 fold by addition of freeze dried CHWPE groups balance the positive amine groups between pH 4.5e5,
up to the 5th day, however; no significant difference (p > 0.05) was hence, the zeta potential decreases to zero. In addition, low NaCl
found between TAC values of S1 and control sample at the 15th day. concentration remains zeta potential high (Ravindran, Williams,
This situation may be explained that binding of polyphenols to milk Ward, & Gillies, 2017). Ayran is an acidic (pH 4.5) and salty (0.5%)
proteins caused to reduce their bioavailability and functionality and dairy beverage. Therefore, its zeta potential was measured
thus to reduce their antioxidant potential. Moreover, the as 3.2 ± 0.32. The interaction between the negatively charged
proteinpolyphenol interaction is observed as maximal at the pH milk proteins and positively charged chitosan might have led to this
of yoghurt (4.6) which is the isoelectric point of the milk proteins. destruction of liposomes. In contrast, presence of maltodextrin in
liposomal powder might prevent from the interaction between
milk proteins and liposomes. As reported before (Thongkaew, Gibis,
Hinrichs, & Weiss, 2014), polysaccharides had the ability of binding
Table 1
Total phenolic content (TPC) (mg GAE/mL), total flavonoid content (TFC) (mg CE/mL), to polyphenols to prevent or decrease polyphenol-protein in-
and total antioxidant capacity (TAC) (TEAC mg/mL) in ayran samples during storage teractions. Hence liposomal stability was maintained. According to
period. these findings, it is possible to say that liposomal encapsulation was
TPC (mg GAE/mL) more effective way to maintain the CHWPE stability in ayran sys-
tem. Moreover; TPC, TFC and TAC of CHWPE was exhibited better
1st Day 5th Day 10th Day 15th Day
results in liposomal powders than liposomal dispersion in ayran
Control 0.27 ± 0.021aA 0.22 ± 0.03aB 0.27 ± 0.04aAC 0.23 ± 0.01aBD during storage.
S1 0.43 ± 0.02bA 0.37 ± 0.01bB 0.36 ± 0.01bB 0.28 ± 0.02aC
S2 0.58 ± 0.03bcA 0.68 ± 0.05cB 0.51 ± 0.03cAC 0.59 ± 0.02bAC
S3 0.60 ± 0.04cA 0.52 ± 0.01dB 0.57 ± 0.06cAC 0.48 ± 0.02cD 3.22. In-vitro bioaccessibility of CHWPE in ayran during storage
S4 1.31 ± 0.04dA 1.23 ± 0.05eA 1.25 ± 0.05dA 1.29 ± 0.03dA period
TFC (mg CE/mL),
TPC, TFC and TAC values of CHWPE were determined at the 1st,
1st Day 5th Day 10th Day 15th Day
Control 0.03 ± 0.003aA 0.02 ± 0.001aB 0.02 ± 0.003aB 0.02 ± 0.001aB
5th, 10th and 15th day in digested ayran samples where the results
S1 0.04 ± 0.004aA 0.03 ± 0.005aB 0.03 ± 0.002aB 0.03 ± 0.004aC showed in Table 2. The bioaccessibility of CHWPE in ayran was
S2 0.12 ± 0.005bA 0.12 ± 0.005bA 0.10 ± 0.02bB 0.03 ± 0.003aC increased when it was added in liposomal powder form instead of
S3 0.06 ± 0.005cA 0.05 ± 0.004cA 0.06 ± 0.003cA 0.05 ± 0.002bA non-encapsulated freeze dried form. Indeed; TPC, TFC and TAC in S1
S4 0.20 ± 0.02dA 0.14 ± 0.01dB 0.14 ± 0.001dB 0.09 ± 0.001cC
did not show a significant difference (p > 0.05) from control ayran
CUPRAC (TEAC mg/mL) after in-vitro digestion during storage period which means that
1st Day 5th Day 10th Day 15th Day unencapsulated CHWPE in ayran was highly degraded after diges-
Control 0.10 ± 0.01aA 0.09 ± 0.01aA 0.10 ± 0.01aA 0.08 ± 0.01aB tion. In terms of TPC, TCF and DPPH assay, bioaccessible CHWPE
S1 0.17 ± 0.01bA 0.17 ± 0.02bA 0.12 ± 0.01aB 0.11 ± 0.01aB was found to be 3 fold higher in both S2 and S3 than S1 at the 1st
S2 0.17 ± 0.03 bA 0.10 ± 0.03acB 0.14 ± 0.03aC 0.11 ± 0.02bBD
day. However, this increment reduced to 2 fold after the 5th day
S3 0.30 ± 0.01cA 0.21 ± 0.02dB 0.17 ± 0.02bB 0.22 ± 0.01cB
S4 0.44 ± 0.04dA 0.22 ± 0.04eB 0.24 ± 0.02cB 0.27 ± 0.02dB where remained up to the end of storage. It was expected that in-
vitro digested CHWPE in S3 would be higher than S2 due to pres-
DPPH (TEAC mg/mL)
ence of chitosan. Several studies reported that covering liposome
1st Day 5th Day 10th Day 15th Day surface with positively charged polymers such as, chitosan, lacto-
Control 0.03 ± 0.005aA 0.02 ± 0.01aA 0.02 ± 0.005aA 0.01 ± 0.005aB
S1 0.05 ± 0.02bA 0.04 ± 0.005bA 0.03 ± 0.005aA 0.01 ± 0.005aB
ferrin or poly(allyl amine) hydrochloride provided stability of li-
S2 0.34 ± 0.02cA 0.23 ± 0.02cB 0.14 ± 0.03bC 0.08 ± 0.01bD posomes in gastro-intestinal track (Agrawal, Harde, Thanki, & Jain,
S3 0.09 ± 0.007dA 0.02 ± 0.001adB 0.02 ± 0.005acB 0.01 ± 0.003acB 2014; Liu, Ye, Liu, Liu, & Singh, 2013). Nevertheless, unstability of
S4 0.43 ± 0.04eA 0.39 ± 0.05eA 0.48 ± 0.01dA 0.25 ± 0.01dB chitosan coated liposomal dispersion in S3, might have affected the
*Ayran with freeze dried cocoa hull waste extract (CHWPE) (S1), ayran with CHWPE bioaccessibility of CHWPE. For this reason, in-vitro digested CHWPE
powder (S2), ayran with chitosan-coated liposomes containing CHWPE (S3), ayran in S2 and S3 might have showed similar increment ratio during
with spray-dried chitosan-coated liposomal powder containing CHWPE (S4), con- storage. On the other hand; TPC, TFC and TAC of CHWPE in S4 were
trol (ayran).
*Values are presented as mean values ± standard deviation (n ¼ 3). Different small
detected to be significantly higher (p < 0.05) than other samples
letters in the columns or different capital letters in the rows represent statistically after in-vitro digestion. Comparing to S1, CHWPE bioaccessibility
significant differences (p < 0.05) in each section. was 5 fold higher in S4 in terms of TPC, TFC and CUPRAC assays.
368 G. Altin et al. / Food Hydrocolloids 81 (2018) 364e370

Table 2
Total phenolic content (TPC) (mg GAE/mL), total flavonoid content (TFC) (mg CE/mL), and total antioxidant capacity (TAC) (TEAC mg/mL) in digested ayran samples during
storage period.

TPC (mg GAE/mL)

1st Day 5th Day 10th Day 15th Day

aA aA aA
Control 0.325 ± 0,0097 0.326 ± 0.008 0.31 ± 0.036 0.32 ± 0.018aA
S1 0.366 ± 0,0064aA 0.347 ± 0.005aA 0.324 ± 0.029aA 0.333 ± 0.024aA
S2 0.936 ± 0,0886bA 0.777 ± 0.039bA 0.76 ± 0.027bA 0.802 ± 0.033bA
S3 0.805 ± 0,0212bA 0.691 ± 0.019cB 0.72 ± 0.038bB 0.731 ± 0.04cB
S4 2.014 ± 0,1165cA 1.632 ± 0.064dB 1.71 ± 0.15cB 1.56 ± 0.022dB

TFC (mg CE/mL)

1st Day 5th Day 10th Day 15th Day

Control 0.013 ± 0.003aA 0.013 ± 0,003aA 0.009 ± 0.003aB 0.007 ± 0.002aB
S1 0.015 ± 0.003aA 0.013 ± 0.003aB 0.011 ± 0.004aB 0.010 ± 0.003aC
S2 0.038 ± 0.006bA 0.031 ± 0.006bB 0.026 ± 0.003bB 0.024 ± 0.004bC
S3 0.040 ± 0.006bA 0.029 ± 0.002cB 0.021 ± 0.005cB 0.019 ± 0.005cC
S4 0.074 ± 0.031cA 0.067 ± 0.021dB 0.05 ± 0.012dB 0.047 ± 0.015dC

CUPRAC (TEAC mg/mL)

1st Day 5th Day 10th Day 15th Day

Control 0.041 ± 0.0073aA 0.039 ± 0.019aAB 0.042 ± 0.023aB 0.041 ± 0.012aC
S1 0.043 ± 0.0062aA 0.041 ± 0.016aA 0.046 ± 0.032aA 0.043 ± 0.018aB
S2 0.167 ± 0.008bA 0.135 ± 0.022bA 0.148 ± 0.007bA 0.141 ± 0.017bB
S3 0.161 ± 0.02bA 0.115 ± 0.014cA 0.142 ± 0.053bB 0.119 ± 0.022cB
S4 0.234 ± 0.04dA 0.218 ± 0.075dB 0.215 ± 0.052cB 0.203 ± 0.023bdC

DPPH (TEAC mg/mL)

1st Day 5th Day 10th Day 15th Day

Control 0.012 ± 0.005aA 0.01 ± 0.006aB 0.015 ± 0.01aB 0.013 ± 0.004aB
S1 0.015 ± 0.002aA 0.014 ± 0.004aA 0.013 ± 0.007aA 0.013 ± 0.005aA
S2 0.036 ± 0.014bA 0.031 ± 0.01bA 0.033 ± 0.051bA 0.025 ± 0.023bB
S3 0.037 ± 0.014bA 0.034 ± 0.016bB 0.034 ± 0.024bB 0.031 ± 0.018cB
S4 0.083 ± 0.008cA 0.08 ± 0.05cA 0.082 ± 0.042cA 0.081 ± 0.043dB

*Ayran with freeze dried cocoa hull waste extract (CHWPE) (S1), ayran with CHWPE powder (S2), ayran with chitosan-coated liposomes containing CHWPE (S3), ayran with
spray-dried chitosan-coated liposomal powder containing CHWPE (S4), control (ayran).
*Values are presented as mean values ± standard deviation (n ¼ 3). Different small letters in the columns or different capital letters in the rows represent statistically sig-
nificant differences (p < 0.05) in each section.

According to DPPH assay, TAC of bioaccessible CHWPE in S4 was difference (p > 0.05) during shelf life period. The bioaccessibility of
found to be 6 fold higher than S1. Meanwhile; TPC, TFC and TAC of catechin was 53%, 48%, 44% and 50% while bioaccessibility of ferulic
CHWPE did not show significant difference (p > 0.05) in in-vitro acid was 83%, 88%, 88% and 82% at the 1st, 5th, 10th and 15th days,
digested S4 during storage since presence of MD provided a stabile respectively. At the end of the storage period, vanilin showed
structure during in-vitro digestion in liposomal powder. Malto- minimum bioaccessibility in S4 (19%), in S3 (16%) and in S1(10%),
dextrin which supported a secondary layer on liposomes other than while p-coumaric acid showed the lowest bioaccessibility (10%) in
chitosan might have enhanced phenolic stability in gastrointestinal S2. This situation may be explained by the suggestion that chemical
tract (Altin et al., 2018), Therefore, CHWPE degradation was pre- structure of phenolic compounds and their location in liposomal
vented from in-vitro digestion due to this carbohydrate matrix re- systems might have affected phenolic degradation level before and
ported by Schrammet et al. (2003); Saura-Calixto, Serrano, and after in-vitro digestion.
Goni (2007) before.

3.33. Quantification and identification of CHWPE in ayran during 4. Conclusion

storage period
In conclusion, liposomal systems in dispersion and powder form
Phenolic profiles of ayran samples before and after in-vitro were employed for the delivery of cocoa hull waste phenolics via
digestion were given in Table 3 and Table 4, respectively. The ayran. It was observed that stability of CHWPE in ayran samples
amount of detected individual phenolics in each sample was showed better results in liposomal powder form before and after
significantly different (p < 0.05) before and after in-vitro digestion. in-vitro digestion regarding total phenolics, total flavonoids and
In freeze dried CHWPE, ten major phenolic compounds, namely, antioxidant capacity even during storage period of 15 days. In
catechin, epicatechin, quercetin, ferulic acid, gallic acid, p-coumaric addition, each individual phenolic compound of CHWPE in lipo-
acid, syringic acid, trans-cinnamic acid, vanilic acid and vanillin somal powder showed the highest stability before and after in-vitro
were detected before and after in-vitro digestion (Altin et al., 2018). digestion. To suggest the form of liposomal system to be used in
Each phenolic compound of CHWPE in S4 showed the highest liquid foods, physicochemical properties of such foods including
stability before and after in-vitro digestion. The findings showed pH, zeta potential etc. will be crucial. Therefore, it is possible to say
that dryingof liposomal systems might have protected phenolic that the use of liposomal delivery systems in powder form should
compounds from interaction with other ayran components like be recommended even in dispersion foods like ayran. Our results
proteins, reducing the risk of phenolic degradation. In S4, the bio- can be guide for studies on functional food development and nu-
accessibility of catechin and ferulic acid showed no significant traceutical field.
 G. Altin et al. / Food Hydrocolloids 81 (2018) 364e370 369

Table 3
Phenolic profile (mg/100 g) of S1 (ayran with freeze dried CHWPE), S2 (ayran with CHWPE powder), S3 (ayran with chitosan-coated liposomes containing CHWPE), S4 (ayran
with spray-dried chitosan-coated liposomal powder containing CHWPE), control ayran during storage period.

Sample Catechin Epicatechin Quercetin Ferulic Acid Gallic Acid p-Coumaric Acid Syringic Acid Transcinnamic Acid Vanilic Acid Vanilin

DAY 1

Control e e e e e e e e e e
S1 12 ± 0.12a 66 ± 1a 26 ± 0.27a 8 ± 0.1a 27 ± 0.2a 87 ± 0.2a 88 ± 0.4a 14 ± 0.2a 15 ± < 0.1a 0.2 ± <0.01a
S2 8 ± 0.1b 68 ± 1.2a 84 ± 1b 67 ± 0.1b 121 ± 0.4b 105 ± 1.9b 91 ± 2.4b 14 ± 0.2a 31 ± 0.8b 1 ± <0.01b
S3 17 ± 0.1c 104 ± 2.8b 57 ± 0.5c 10 ± 0.1c 41 ± 0.3c 121 ± 0.5c 87 ± 0.2ac 17 ± 0.1b 20 ± < 0.1c 0.2 ± <0.01ac
S4 33 ± 0.11d 334 ± 1c 193 ± 1d 64 ± 1.8d 177 ± 4.5d 396 ± 3.9d 195 ± 0.3d 74 ± 0.5c 197 ± 0.2d 0.4 ± 0.03d

DAY 5

Control e e e e e e e e e e
S1 5 ± 0.2a 55 ± 1.1a 29 ± 0.5a 7 ± 0.2a 19 ± 0.4a 81 ± 0.7a 81 ± 2.3a 12 ± 0.1a 16 ± 0.2a 1 ± <0.01a
S2 8 ± 0.1b 67 ± <0.1b 91 ± 0.8b 69 ± <0.1b 120 ± 0.2b 124 ± 0.6b 81 ± 0.3a 16 ± 0.2b 18 ± 0.7b 3 ± <0.01b
S3 16 ± 0.4c 118 ± 2.1c 50 ± 0.8c 10 ± 0.01c 42 ± 0.4c 107 ± 0.8c 83 ± 0.5b 14 ± 0.1c 21 ± 0.16c 0.2 ± <0.01c
S4 60 ± 1.9d 330 ± 1.1d 137 ± 4.0d 62 ± 0.1d 170 ± 2.7d 339 ± 1d 161 ± 2.3c 56 ± 0.3d 192 ± 0.6d 1 ± < 0.01d

DAY 10

Control e e e e e e e e e e
S1 5 ± < 0.1a 39 ± < 0.1a 24 ± 0.8a 6 ± 0.2a 30 ± 0.1a 75 ± 0.4a 46 ± 0.1a 13 ± 0.2a 11 ± 0.2a 0.1 ± <0.01a
S2 7 ± 0.1b 65 ± 0.1b 94 ± 0.1b 59 ± 0.2b 112 ± 0.1b 118 ± 1b 32 ± 0.1b 16 ± 0.1b 21 ± 0.5b 2 ± <0.01b
S3 19 ± 0.3c 115 ± 1.3c 60 ± 0.4c 13 ± 0.1c 43 ± 0.1c 105 ± 0.1c 94 ± 0.5c 22 ± 0.3c 31 ± 0.2c 3 ± <0.01c
S4 49 ± 0.1d 219 ± 2.7d 195 ± 0.5d 59 ± 0.8d 163 ± 0.1d 360 ± 0.6d 151 ± 0.2d 61 ± 0.4d 189 ± 0.3d 2 ± 0.01d

DAY 15

Control e e e e e e e e e e
S1 5 ± 0.1a 38 ± 0.1a 18 ± 1a 2 ± 0.2a 15 ± 0.1a 47 ± 0.4a 40 ± 0.1 7 ± 0.1a 9 ± 0.2a 0.1 ± <0.01a
S2 10 ± 0.4b 47 ± 0.2b 54 ± 1.6b 50.2 ± 0.1b 94 ± 0.1b 110 ± 0.1b 34 ± 0.1 22 ± 0.3b 22 ± 0.3b 0.3 ± <0.01b
S3 13 ± 0.1c 119 ± 0.6c 61 ± 0.5c 10 ± 0.1c 49 ± 0.4c 102 ± 0.8c 85 ± 0.2 17 ± 0.1c 29 ± 0.1c 0.2 ± <0.01c
S4 36 ± 0.2d 104 ± 1d 108 ± 5d 42 ± 0.1d 187 ± 2.6d 388 ± 3.9d 132 ± 3.1 56 ± 1.4d 181 ± 0.7d 5 ± 0.3d

*Values are presented as mean values ± standard deviation (n ¼ 3). Different small letters in the columns represent statistically significant differences (p < 0.05) in each
section.

Table 4
Phenolic profile (mg/100 g) of S1 (freeze dried CHWPE), S2 (ayran with CHWPE powder), S3 (ayran with chitosan-coated liposomes containing CHWPE), S4 (ayran with spray-
dried chitosan-coated liposomal powder containing CHWPE), control ayran during storage period after in-vitro digestion.

Sample Catechin Epicatechin Quercetin Ferulic Acid Gallic Acid p-Coumaric Acid Syringic Acid Trans-Cinnamic Acid Vanilic Acid Vanilin

DAY 1

Control e e e e e e e e e e
S1 3.0a 24.9a 18.8a 4.4a 14.5a 14.4a 10.0a 6.9a 11.9a <0.001a
S2 6.2b 72.3b 79.9b 50.9b 116.9b 125.9b 100.7b 4.2b 33.3b 0.8b
S3 5.2c 91.8c 39.1c 10.8c 36.3c 37.3c 20.5c 11.8c 22.6c 0.1c
S4 17.7d 326.5d 123.8d 52.9d 131.8d 169d 96.1d 75.7d 70.2d 0.3d

DAY 5

Control e e e e e e e e e e
S1 2.9a 41.8a 17.2a 4.4a 11.3a 12.7a 8.9a 6.3a 7.7a <0.001a
S2 5.0b 63.9b 66.7b 42.1b 108.3b 97.8b 69.8b 6.2b 9.4b 0.7b
S3 4.3c 75.9c 31.7c 10.0c 20.1c 20.9c 18.1c 10.0c 20.6c 0.1c
S4 29.1d 266.8d 170.1d 56.7d 146.3d 157.6d 85.9d 67.3d 93.5d 0.5d

DAY 10

Control e e e e e e e e e e
S1 3.0a 23.0a 15.9a 3.6a 10.7a 12.5a 6.6a 6.2a 9.4a <0.001a
S2 4.8b 60.3b 65.4b 42.9b 106.1b 104b 21.6b 9.5b 8.7b 3b
S3 8.3c 89.8c 31.9c 13.3c 22.3c 24.5c 18.5c 13.2s 0.20c 1c
S4 21.7d 365.5d 174.5d 52.2d 146.2d 153.9d 99.1d 56.6d 116.2d 4d

DAY 15

Control e e e e e e e e e e
S1 2.1a 19.8a 10.8a 1.3a 10.3a 12.7a 8.8a 3.2a 1.7a <0.001a
S2 2.9b 39.8b 58.8b 33.2b 10.3b 82.5b 11b 7.7b 9.2b 0.5b
S3 4.1ac 88.1c 32.0c 11.5c 20.4c 10.3 14.2c 13.5c 11.4ac 0.1
S4 18.2d 303.4d 164.9d 50.7d 137.2d 14.1c 98.3d 47.6d 130.7d 0.9d

*Values are presented as mean values ± standard deviation < 0.05 (n ¼ 3). Different small letters in the columns represent statistically significant differences (p < 0.05) in each
section.

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