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Efficacy of Whey Protein Film Incorporated with Portuguese
Green Tea (Camellia sinensis L.) Extract for the Preservation of
Latin-Style Fresh Cheese
João Robalo 1 , Maria Lopes 1 , Olga Cardoso 1,2 , Ana Sanches Silva 1,3,4, * and Fernando Ramos 1,5
1 Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] (J.R.);

[email protected] (M.L.); [email protected] (O.C.); [email protected] (F.R.)
2 Chemical Process Engineering and Forest Products Research Centre, 3030-194 Coimbra, Portugal
3 National Institute for Agrarian and Veterinarian Research (INIAV), I.P., 4485-655 Vairão, Portugal
4 Center for the Study of Animal Science (CECA)—Institute of Sciences, Technologies and
Agroenvironment (ICETA), University of Porto, 4099-002 Porto, Portugal
5 Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and
Technology (REQUIMTE), University of Porto, 4099-002 Porto, Portugal
* Correspondence: [email protected] or [email protected]
Abstract: Fresh cheese composition favors the growth of microorganisms and lipid oxidation, leading
to a short shelf life. Whey protein concentrates can be used to produce active films in which green
tea (Camellia sinensis L.) extract, rich in bioactive compounds, namely catechins, can be incorporated.
Thus, the main objective of this study was to evaluate the efficacy of an edible active film, incorporated
with green tea extract, to preserve goat and mixture (goat and sheep) fresh cheeses. Our results
demonstrated that Portuguese green teas (antioxidant activity coefficient—AAC = 746.7) had superior

antioxidant capacity to that of the evaluated Asian green tea (AAC = 650). Furthermore, green
Citation: Robalo, J.; Lopes, M.; tea produced from the leaves of the new Portuguese Chá Camélia tea plantation had the highest
Cardoso, O.; Sanches Silva, A.; potential to retain the antioxidant capacity (97.3%). Additionally, solid–liquid extractions led to
Ramos, F. Efficacy of Whey Protein extracts with higher antioxidant activity (AAC = 1500), but Soxhlet extractions presented higher
Film Incorporated with Portuguese yield (43%). Furthermore, the active film incorporated with Portuguese green tea extract exhibited a
Green Tea (Camellia sinensis L.)
high antioxidant capacity (AAC ≈ 595.4). In addition, the active film effectively delayed the lipid
Extract for the Preservation of
oxidation of the evaluated fresh cheeses (3.2 mg MDA Eq/kg) when compared with the control
Latin-Style Fresh Cheese. Foods 2022,
(4.2 mg MDA Eq/kg). Moreover, the active films effectively inhibited the growth of microorganisms,
11, 1158. https://doi.org/10.3390/
especially E. coli (1.5 × 10 CFU/g), when compared with the blank (2.2 × 102 CFU/g). This study
foods11081158
suggests that the new whey protein film incorporated with Portuguese green tea extract has the
Academic Editor: Barbara Speranza potential to be used to extend fresh cheese shelf life.
Received: 29 March 2022
Accepted: 13 April 2022 Keywords: active packaging; antimicrobial activity; antioxidant capacity; edible film; fresh cheese;
Published: 16 April 2022 green tea; whey protein
Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil-
1. Introduction
iations.
Polyethylene, polypropylene, and polyamide are the most common polymers used
for cheese packaging; however, legislation concerning the migration of chemicals from
food-contact materials into cheese can restrict the additives used in their formulations. In
Copyright: © 2022 by the authors. addition, the characteristics of these polymers, such as nonbiodegradability and noned-
Licensee MDPI, Basel, Switzerland. ibility, can lead to environmental issues. These significant problems have motivated the
This article is an open access article research community to search for new packaging solutions for cheese [1]. The cheese
distributed under the terms and industry faces yet another challenge, as fresh cheeses (classified as unripened cheeses) are
conditions of the Creative Commons a very perishable food product. According to the samples used in this research, when
Attribution (CC BY) license (https://
stored properly, in refrigerated temperatures (0–7 ◦ C) in an unopened package, fresh cheese
creativecommons.org/licenses/by/
can last between 8 and 12 days. However, after opening the package, fresh cheese is
4.0/).
Foods 2022, 11, 1158. https://doi.org/10.3390/foods11081158 https://www.mdpi.com/journal/foods

Foods 2022, 11, 1158 2 of 23
acceptable for consumption for 1–2 days. Because of their high pH (>5) and water activity,
fresh cheeses are particularly vulnerable to postprocess microbial contamination [2–5].
During handling and storage, fresh cheese is vulnerable to the growth of nonpathogenic
and pathogenic microorganisms. The growth of bacteria, yeast, and molds may not only
reduce their quality but affect their safety, since colonization by pathogenic bacteria, such as
Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes, may endanger
consumers by causing foodborne diseases [1,6,7]. In addition, lipid oxidation can alter
the type and concentration of certain chemical compounds in cheese, altering the cheese’s
nutritional value and causing the loss of liposoluble vitamins and organoleptic properties,
resulting in unpleasant tastes and/or aromas and shortening the shelf life (i.e., the length
of time during which the food maintains its acceptable or desirable characteristics under
specified storage and handling) of the food [8].
Active edible films are edible polymeric materials incorporated with biologically active
agents, such as antioxidants or antimicrobials, able to reduce the risk of foodborne bacteria
growth and fungal contamination or inhibit oxidation, allowing improvement of packaged
foods’ shelf life and quality [9–11]. Whey protein is a nutrient-rich by-product of the cheese
industry that can be processed into whey protein concentrate (WPC) powders, which can be
used as film-forming biopolymers in edible packaging [1,12]. Whey protein has already been
applied to edible coatings and film formulations developed for unripened cheeses [13–16].
Whey protein-based edible films have exhibited not only good mechanical properties, com-
pared with those of standard synthetic polymer films, but good barrier properties against
aromas, lipids, and oxygen. However, their hydrophilic nature limits their capacity to act
as barriers against water vapor [17,18]. Plasticizers (e.g., glycerol—E 422) can be added to
surpass this limitation. This way, whey protein-based films obtain better moisture resistance
and improve their resilience (extensibility and flexibility) [17]. Additionally, whey proteins can
be obtained from renewable resources and degrade faster than other polymeric materials [10].
To be effective, a whey protein-based edible film requires, besides a polymeric matrix, an
active agent. One of the latest active packaging trends is using natural plant extracts as
antioxidant and antimicrobial agents [13,19]. Within natural plants extracts, green tea (Camellia
sinensis L.) extract, because of its physicochemical composition, is a promising antioxidant
and antimicrobial agent to be incorporated in edible packaging intended to potentially extend
fresh cheese shelf life.
Green tea contains, predominantly, caffeine, a known central nervous system stimu-
lant; vitamins; carbohydrates; amino acids; and polyphenols, such as flavonoids, antho-
cyanins, and phenolic acids [20–22]. Polyphenols are phytochemicals that have drawn
increasing scientific attention because of their potential therapeutic effects against a wide
variety of diseases [23]. Notably, during the green tea manufacturing process, polyphenol
oxidase activity is inactivated, preventing any oxidation reaction from occurring. There-
fore, when compared with other types of teas, green tea contains much higher amounts
of phenolic compounds, since it maintains not only its original structure but its overall
composition [21,24]. Composed essentially by polyphenols, mainly catechins (including epi-
catechin, epigallocatechin, epigallocatechin-gallate (EGCG), and epicatechin-gallate), and
caffeine (2.5–4% dry weight), green tea extracts may be natural antioxidant agents [25–27].
Moreover, green tea extracts and EGCG have been shown to have the capacity to suppress
foodborne pathogens such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes,
Salmonella typhimurium, Clostridium perfringens, Pseudomonas aeruginosa, Pseudomonas fragi,
Helicobacter pylori, and Brochothrix thermosphacta and therefore may be natural antimicrobial
agents as well [6]. This actively demonstrates that green tea extracts may act as antioxidant
and antimicrobial agents when incorporated into biopolymeric matrices such as whey. For
instance, Castro et al. [17] and Andrade et al. (2019) [8] developed whey protein-based
films incorporated with green tea extracts and applied them to salmon and salami, respec-
tively, to inhibit lipid oxidation. Portugal has, to date, three tea plantations, Gorreana,
Chá Camélia, and Chá Porto Formoso, among which Gorreana and Chá Camélia produce
outstanding green teas that can be used to obtain green tea extracts. Gorreana currently
Foods 2022, 11, 1158 3 of 23
has the largest tea plantation in Europe, and it is internationally recognized not only for the
quality and uniqueness of its teas (black, green, and oolong) but also for the production of
100% organic, chemical-free teas, as the use of synthetic chemical pesticides is not justified
given that the island’s climate does not allow the development of tea plant pests. Gorreana
produces and markets three varieties of green tea, namely “Encosta de Bruma”, “Pérola”,
and “Hysson”. The latter variety was selected as a sample in this study. The Chá Camélia
plantation was born on the northern Portuguese coast and is the only producer of artisanal,
organic, Asian-style green tea in mainland Portugal; this tea was used as a sample in this
study. In addition, it produces “Florchá”, which consists of dehydrated flowers of Camellia
sinensis L. that are used to make delicate, caffeine-free herbal infusions, which were also
used as a sample in this study. Finally, the Porto Formoso tea factory, located on the north
coast of the island of São Miguel, currently produces black tea of the following varieties:
“Orange Pekoe”, “Pekoe”, “Broken Leaf”, and “Azores Home Blend”. However, none of
these varieties were selected for this study.
A wide variety of edible films and coatings have been developed for multiple types of
cheese [1]. However, according to the bibliography, only six edible packages have been de-
veloped to date. In addition, even though multiple polymeric matrices (cassava starch [28],
liquid whey protein concentrate [13], chitosan [29], chitosan plus whey protein [15], and
furcellaran plus whey protein isolate [14,16]) and active compounds (yerba mate and
white tea [14], green tea and Pu-erh [16], Lactobacillus acidophilus [28], Chinese cinnamon
bark [13], and bacteriocins [29]) have been used to develop active packages for several
unripened types of cheese (fresh soft rennet-curd cheese [14], quark [16], Manaba fresh
white cheese [28], ricotta cheese [15], Eastern European curd cheese [13], and Colombian
fresh cheese [29]), no scientific findings are yet available on edible films’ application to and
effect on Latin-style fresh cheese, a type of fresh cheese produced by enzymatic coagulation
of milk with rennet, without adding starter cultures, traditionally made in the Iberian
peninsula from pasteurized goat or cow milk. Latin-style fresh cheeses do not require the
addition of starter cultures and therefore present high pH values (>5.0). In addition to hav-
ing low salt and high moisture content, they are usually made without preservatives. These
characteristics favor the growth of microorganisms and reduce the cheeses’ shelf life [3,7].
Therefore, it is interesting to evaluate the effect of a whey protein film incorporated with
green tea extract on the preservation of this type of fresh cheese, as it is a nonripened cheese
widely consumed in the Iberian peninsula (particularly in Portugal) and very perishable,
demanding consumption shortly after production.
In this context, the present research study’s main objective was to characterize and
compare the antioxidant properties of Portuguese (Gorreana and Chá Camélia) and Asian
(Happy Flora) green teas, including Chá Camélia green tea from 2020 production; optimize
the preparation of green tea extract; and evaluate the efficacy of an edible active film
incorporated with green tea extract in preserving goat and mixed (goat and sheep) fresh
cheeses. This packaging incorporated Portuguese green tea extract to potentially extend
Latin-style fresh cheese’s shelf life. In addition, this was the first study evaluating the
antioxidant capacity of Chá Camélia, the first green tea from continental Portugal.
2. Materials and Methods

2.1. Green Tea Samples
In this study, three Portuguese samples from C. sinensis L. were used, a green tea sample
from the oldest tea plantation in Europe, called Gorreana (37◦ 490 06.700 N 25◦ 240 09.100 W), and
two samples from the first tea plantation in continental Portugal, Chá Camélia (41◦ 190 56.800 N
8◦ 380 31.200 W). The latter samples were produced according to traditional organic farming
methods. From the Gorreana plantation, located on the island of São Miguel (Azores), the
“Hysson” variety was chosen. This tea is produced after harvesting, in July and August,
from the first three leaves of C. sinensis. From the Chá Camélia plantation, located on the
northern Portuguese coast, two samples were evaluated. One was obtained from the leaves
of C. sinensis from the first green tea harvest, Asian style, in continental Portugal, and the
Foods 2022, 11, 1158 4 of 23
other was produced in autumn, resulting from the dehydration of flowers of the tea plant (C.
sinensis). Furthermore, an Asian tea marketed by ADP, LDA (39◦ 260 55.2” N 8◦ 470 20.4” W)
as Happy Flora was selected. The Gorreana-brand and Happy Flora brand green teas were
purchased locally in a commercial area in Coimbra. The samples from the Chá Camélia brand
(green tea and C. sinensis flowers) were kindly supplied by the company.
2.2. Preparation of Green Tea Infusions

The teas were prepared as traditionally as possible. Two hundred milliliters of tap
water was heated to 75 ◦ C, and immediately afterward, 2 g of C. sinensis leaves/flowers,
weighed previously, was soaked for 3 min, with the aid of a tea filter, in the absence of a
sachet. At this stage, for each variety, 4 infusions were prepared. The 1st infusions were
prepared using dry leaves of different varieties of green tea, which were designated as G
(Gorreana), H (Happy Flora), CA (Chá Camélia—leaves), and CB (Chá Camélia—flowers).
Then, the 2nd, 3rd, and 4th infusions were prepared sequentially from the reuse of the
same leaves/flowers, these being designated by *2, *3, and *4, respectively (* corresponds
to G/H/CB/CA). Finally, the antioxidant capacity, total phenolic content (TPC), and total
flavonoid content (TFC) of the infusions and teas were evaluated. The retention potential
of the samples was evaluated by comparing the antioxidant properties, TPC, and TFC of
the 4th infusion with those of the 1st infusion of the same sample.
2.3. Extraction of Green Tea Extracts

2.3.1. Conventional Solid–Liquid Extraction
Initially, the C. sinensis samples were ground, homogenized, and sieved. Five grams of
each sample (AS 220.R1 PLUS scale) was subjected to solid–liquid extraction with 50 mL
of absolute ethanol (99.8%) and were placed in a horizontal shaker (Edmund Bühler KL2)
for 30 min at 400 rpm, followed by centrifugation (Sigma 3–16 K centrifuge, Osterode,
Germany) at 5000× g for 15 min at 15 ◦ C. Afterward, the supernatant was removed into
a pyriform flask, and using a rotary evaporator, the ethanol was evaporated entirely at
40 ◦ C. Next, the extract was removed from the flask using a spatula and stored in a closed
container at 5 ◦ C. Then, a solution was prepared using 10 mg of the extract and 10 mL
of ethanol. Finally, the antioxidant capacity was evaluated. The yield of the solid–liquid
extraction was calculated using the following equation:
A−B
Yield (%) = × 100. (1)
C
where A represents the weight of the extraction flask with extract, B represents the weight
of the empty extraction flask, and C represents the amount of tea sample used to perform
the extraction.
2.3.2. Solid–Liquid Extraction with Soxhlet Apparatus

Five grams of each sample was weighed into a Soxhlet cartridge, which was placed
in a Soxhlet extractor using 150 mL of absolute ethanol (99.8%) as solvent for 6 h. Using a
rotary evaporator, the solvent was completely evaporated at 40 ◦ C. Next, the dry extract
was removed from the distillation flask using a spatula and stored in a closed container
at 5 ◦ C. Afterward, a solution was prepared using 10 mg of the dry extract and 10 mL of
ethanol to allow the evaluation of the antioxidant capacity. The yield of the solid–liquid
extraction via Soxhlet apparatus was calculated using Equation (1) as previously described.
2.4. Preparation of Whey-Based Protein Films

The method of preparing whey protein films was adapted from Ribeiro-Santos et al. [30].
Initially, ultrapure water was added to the whey protein concentrate (acquired online from
Myprotein® —“impact whey protein”). Then, homogenization was carried out using a mag-
netic stirrer, obtaining a solution (8%, w/w protein). The pH was, when necessary, adjusted
to 7.0 using 1M NaOH. Subsequently, the protein was denatured by heating the solution in
Foods 2022, 11, 1158 5 of 23
a thermostatic bath for 30 min at 80 ◦ C. The solution was rapidly cooled in an ice bath to
room temperature (+/−23 ◦ C). Glycerol 1:1 (protein–glycerol) was added and mixed into
the solution. Furthermore, 2.5% (w/w) of the extract was incorporated into the formulation
and then homogenized using an Ultra-Turrax at 14,000 rpm for 2 min. The solution was
distributed in Petri dishes (13.5 mL/100 cm2 ). Finally, the Petri dishes were incubated in an
oven (Memmert 854, Schwabach, Germany) at 40 ◦ C for 24 h. A film without incorporation
of extract in the film-forming solution was used as a blank (control). The films were stored
in the Petri dishes at refrigerated temperatures. To enable the antioxidant evaluation of the
films, a migration assay was performed; 6 cm2 of each film (blank and active) was immersed
into 10 mL of ethanol (95%) and incubated at 40 ◦ C for 10 days. After the incubation, the
antioxidant capacity was immediately evaluated.
2.5. Evaluation of the Antioxidant Properties

Before evaluation of antioxidant properties, green tea extract (dry powder format) was
diluted in ethanol.
2.5.1. β-Carotene Bleaching Assay

The β-carotene bleaching test allows evaluating antioxidant effects against lipid per-
oxidation [31]. Specifically, when linoleic acid is in the presence of reactive oxygen species
(ROS) or oxygen, a peroxyl radical is formed (LOO•). This radical forms a stable β-carotene
radical by reacting with β-carotene, reducing the amount of β-carotene. However, in the
presence of an antioxidant, a competitive reaction occurs between the antioxidant and
β-carotene with the peroxyl radical [32]. This reaction occurs in an aqueous emulsion of
linoleic acid prepared using a phase stabilizer, namely Tween® 40 (Sigma-Aldrich; Madrid,
Spain) [33]. Under standard settings, thermal induction (50 ◦ C) results in the oxidation
of the fatty acid-generating radicals, which causes the discoloration of the yellow-colored
emulsion [34,35]. However, in the presence of an antioxidant, this discoloration can be
delayed via the previously mentioned competitive reaction. These antioxidant effects can be
quantified through spectrophotometry (470 nm) by measuring the rate at which β-carotene
absorbance decays [32,34].
The procedure of this assay followed the method described by Miller and adapted
by Andrade et al. [36]. Initially, a solution was prepared by dissolving β-carotene in
chloroform (0.2 mg/mL). Next, an emulsion of β-carotene and linoleic acid was prepared
using 20 mg of linoleic acid, 200 mg of Tween® 40, and 1 mL of the previously prepared
solution. Chloroform was evaporated at 40 ◦ C on a rotary evaporator. Thereafter, 50 mL of
ultrapure water was added, and the solution was vigorously stirred. After it was prepared,
5 mL of the emulsion was added to 200 µL of the sample. The samples were kept in a
Gerhardt SV24 thermostatic bath (1500 w) at 50 ◦ C for 120 min. Using a Hitachi U-3900
spectrophotometer (Hitachi, Tokyo, Japan), the absorbances of the samples were measured
at 470 nm. The antioxidant activity coefficient (AAC) was calculated using Equation (2):
AS − AC2
AAC = × 1000. (2)
AC0 − AC2
where AS represents the absorbance of the samples, AC0 represents the absorbance of the
control before heating, and AC2 represents the absorbance of the control after heating.
2.5.2. DPPH Radical-Scavenging Method

The DPPH radical (2,2-diphenyl-1-picryl-hydrazyl) assay is an easy and quick method
that allows evaluating a sample’s capability to inhibit lipid oxidation by determining its free
radical-scavenging capacity [37–39]. In short, the antioxidant reacts with the radical DPPH•,
reducing it to diphenylpicrylhydrazine, leading to the discoloration of the solution [33].
Briefly, 2 mL of a methanolic solution of DPPH (14.6 µg/mL) was added to 50 µL
of the sample. After homogenization, the solutions were protected from light for 30 min.
Foods 2022, 11, 1158 6 of 23
Absorbance was measured at 515 nm using a Hitachi U-3900 spectrophotometer(Tokyo,

Japan. DPPH• inhibition percentage (IP%) was calculated using Equation (3):
AC − AS
IP (%) = × 100. (3)
AC
where AC represents the absorbance of the control and AS represents the absorbance of
the sample. The method applied followed the method described by Blois (1958) [40] with
some adaptations. A calibration curve (y = 0.6051x + 7.0233, r2 = 0.9979) was drawn up by
plotting different concentrations of Trolox (5–150 µg/mL) in order to express the results in
Trolox equivalent (TE). Results were expressed as µg Trolox equivalent/g or mL of sample.
2.5.3. Determination of Total Flavonoid Content (TFC)

The spectrophotometric assay, based on the production of aluminum complexes and
their spectrophotometric determination, is one of the most widely used procedures for
determining total flavonoid content [41]. According to the basic concept of the aluminum
chloride (AlCL3 ) colorimetric method, AlCl3 forms acid-stable complexes with the C-4 keto
group and either the C-3 or C-5 hydroxyl group of flavones and flavonols, resulting in a
yellow color [42–44].
To determine the TFC, the method described by Yoo et al. [45] was applied. Briefly,
4 mL of ultrapure water was added to 1 mL of sample. Then, 300 µL of an aqueous solution
of sodium nitrite (5%, w/v) and the mixture were homogenized. After 5 min, 600 µL of an
aqueous aluminum chloride solution (10%, w/v) was added, and the solution was homoge-
nized again. After 6 min, 2 mL of an aqueous solution of sodium hydroxide (1 M, w/v) and
2.1 mL of ultrapure water were added. All steps occurred at room temperature. Finally,
the samples were homogenized, and the absorbance was measured at 510 nm using a Hi-
tachi U-3900 Spectrophotometer (Tokyo, Japan). A calibration curve (y = 0.0027x − 0.0017,
r2 = 0.9972) was built for the TFC assay by plotting different concentrations of epicatechin
(5–200 µg/mL) in order to express the results in epicatechin equivalent (ET). Results were
expressed in mg epicatechin equivalent/g or mL of sample.
2.5.4. Determination of Total Phenolic Compounds (TPC)

The Folin–Ciocâlteu reagent (FCR) assay is commonly used to determine total phe-
nolic content (TPC). This colorimetric assay of phenolic and polyphenolic antioxidants is
conducted with Folin’s phenol reagent, a combination of phosphomolybdate and phos-
photungstate. The FCR assay is based on the single electron transfer (SET) from phenolic
chemicals to FCR in an alkaline solution resulting in a blue-colored chromophore that can
be detected spectrophotometrically at 750–765 nm. Specifically, phenolics are energetically
oxidized, yielding O2 , which combines with molybdate to yield colored molybdenum ions,
MO4+ [33,46].
The content of TPC was determined by the method described by Singleton and Rossi
(1965) [47]. Briefly, 1 mL of each sample was mixed with 7.5 mL of FCR (10%, v/v). After
5 min, 7.5 mL of a 60 mg/mL (w/v) aqueous sodium carbonate solution was added. After
homogenization, the solutions were kept in the dark for 120 min at room temperature.
Finally, absorbance was measured using a Hitachi U-3900 spectrophotometer (Tokyo, Japan
at 725 nm. A gallic acid calibration curve (y = 0.0064x + 0.0463, r2 = 0.9929) was built by
plotting different concentrations of gallic acid (5–150 µg/mL) in order to express the results
in gallic acid equivalent (GAE). Results were expressed in mg gallic acid equivalent/g or
mL of sample.
2.6. Fresh Cheese Samples

For the microbiological analysis, goat fresh cheese and a mixture fresh cheese (a cheese
obtained from a mixture of sheep and goat milk) were acquired at a local cheese factory
(Queijaria da Licínia, Rabaçal, Portugal ), while for the lipid oxidation analysis, goat and
mixed fresh cheeses were purchased at a commercial store in Coimbra (Portugal). In
Table 1. Nutrition declaration per 100 g of goat fresh cheese and mixture fresh cheese samples.
Nutrition Declaration per 100 g of Product
Goat Fresh Cheese Mixture Fresh Cheese
(Queijaria Licínia, Rabaçal, Portugal)
Energy (kJ)–Energy (kcal) 10.39–259 10.82–261
Foods 2022, 11, 1158 Fat (g) 18.8 20.8 7 of 23
Of which saturated fatty acids (g) 12.98 13.93
Carbohydrates 4.2 4.6
Of which sugars 0.5 0.9
both situations, after the acquisition or purchase, the fresh cheeses were, on the same day,
Protein (g) 16.6 13.8
packaged with both the active and the control films (Figure 1) and stored at 5 ◦ C. The
Salt (g) 1.10 0.55
nutritional compositions of the samples can 45.82
Water content (g)
be consulted in Table 1. After
45.42
a week in storage,
microbiological analysis and lipid oxidation evaluations were performed.
A B
FigureFigure
1. Mixture fresh cheeses
1. Mixture packaged
fresh cheeses with the
packaged withactive film (A)
the active filmand
(A)the
andcontrol film (B).
the control film (B).
2.7. Evaluation of the Lipid

Table 1. Nutrition Oxidation
declaration perStatus
100 g of(TBARS Assay)
goat fresh cheese and mixture fresh cheese samples.
The antioxidant capacity of active packaging, when in contact with two types of fresh
cheese Nutrition
(preparedDeclaration
with goat per
milk100 g ofwith
and Product
goat/sheep
Goatmilk
Freshmixture)
Cheese purchased
Mixtureat a com-
Fresh Cheese
(Queijaria Licínia, Rabaçal, Portugal)
mercial store, was analyzed through the TBARS assay. The two types of cheese were
wrapped with theEnergy (kJ)–Energy
edible (kcal)
active films and with the control 10.39–259
films (without green 10.82–261
tea extract)
and stored at 5 °C to simulate
Fat (g) standard storage conditions. 18.8After one week of storage
20.8 at
refrigeratedOf which saturated
temperatures, thefatty
assayacids
was(g)carried out. Briefly, 10 mL of trichloroacetic acid
12.98 13.93
Carbohydrates 4.2 4.6
Of which sugars
0.5 0.9
Protein (g) 16.6 13.8
Salt (g) 1.10 0.55
Water content (g) 45.82 45.42
2.7. Evaluation of the Lipid Oxidation Status (TBARS Assay)

The antioxidant capacity of active packaging, when in contact with two types of
fresh cheese (prepared with goat milk and with goat/sheep milk mixture) purchased at a
commercial store, was analyzed through the TBARS assay. The two types of cheese were
wrapped with the edible active films and with the control films (without green tea extract)
and stored at 5 ◦ C to simulate standard storage conditions. After one week of storage at
refrigerated temperatures, the assay was carried out. Briefly, 10 mL of trichloroacetic acid
(7.5%, v/v) was mixed with 5 g of each sample and homogenized in a compact stirrer for
1 h at 350 rpm. Then, the samples were filtered through a Whatman number 1 paper filter.
Afterward, 5 mL of thiobarbituric acid (2.88 mg/mL, w/v) (TBA) was mixed with 5 mL of
the filtrate. Then, the samples were submitted to 95 ◦ C in a heat block for 30 min and then
rapidly cooled in ice for 15 min. The absorbance was measured, using a Hitachi U-3900
spectrophotometer (Tokyo, Japan), at 530 nm against the blank assay (5 mL of water and
5 mL of TBA). For this assay, a calibration curve (y = 17.657x − 0.0326; r2 = 0.9905) was
built by plotting different concentrations of 1,1,3,3-tetramethoxypropane (10–55 µg/mL) to
express the results in mg of malonaldehyde equivalent per kg of sample (mg MDA Eq/kg).
2.8. Microbiological Analysis

The antimicrobial capacity of active packages in contact with two types of fresh cheese
(prepared with goat milk and with goat/sheep milk mixture) was analyzed by counting the
microorganisms present in the cheese. To observe the antimicrobial capacity of the active
Foods 2022, 11, 1158 8 of 23
packages, various parameters were determined: total microorganisms at 30 ◦ C according

to ISO 4833-1:2013, total of psychrophiles according to ISO 17410:2019, count of E. coli
according to ISO 16649-2:2001, and count of coagulase-positive staphylococci according to
ISO 6888-1.
The counts of the different microorganisms were carried out on fresh goat cheese and
fresh mixture (sheep and goat) cheese produced on the day. The two types of cheese were
wrapped with the edible active films and with the control films (without green tea extract)
and stored at 5 ◦ C to simulate standard storage conditions. After one week of storage at
refrigerated temperatures, microorganism counts were carried out.
2.9. Statistical Analysis

For statistical analysis, the GraphPad Prism (v9.1.2) software developed by Dr. Harvey
Motulsky (San Diego, California), was used. One-way ANOVAs were performed using a
significance level of 0.05. Additionally, Tukey’s multiple comparison tests were performed.
3. Results and Discussion

3.1. Evaluation of the In Vitro Antioxidant Capacity, TPC, and TFC
In this research study, the antioxidant capacity was evaluated, and the TPC and TFC
determined, of tea infusions from different green tea varieties. Subsequently, the same
evaluations were performed on different green tea extracts. Finally, the antioxidant capacity
was evaluated, and the TPC and TFC determined, of the active edible film.
To assess the antioxidant capacity of the active edible film and green tea infusions and
extracts, DPPH free radical-scavenging and β-carotene bleaching methods were selected
To evaluate TPC, a Folin–Ciocâlteu’s reagent assay was used. To evaluate TFC, a
spectrophotometric assay based on the production of aluminum complexes and their
spectrophotometric determination was used.
3.1.1. Evaluation of the Antioxidant Capacity, TPC, and TFC among Different Green
Tea Infusions
This study evaluated the antioxidant capacity of green tea infusions from two Por-
tuguese brands: Gorreana and Chá Camélia. From the Gorreana tea brand, the “Hysson”
variety was chosen, and from the Chá Camélia brand, one infusion was prepared from
the leaves of Camellia sinensis L., and another from the flowers of C. sinensis. In addition,
the antioxidant capacity of an Asian green tea marketed as Happy Flora was assessed.
Furthermore, the potential of retention of the antioxidant capacity was evaluated through
antioxidant analysis of infusions made by reusing the same leaves/flowers.
In general Figures 2–4 show that infusions from Gorreana green tea had the highest
antioxidant activity and that the analyzed infusions of green tea from the Gorreana (G)
and Chá Camélia (CA) plantations had superior phenolic and flavonoid content than the
analyzed infusion of Asian green tea (H). Furthermore, Figures 2–5 demonstrate that,
overall, tea from the Chá Camélia plantation, obtained from the leaves of C. sinensis (CA),
had the highest potential to retain the antioxidant capacity.
Figures 3–5 corroborate the previously established assumptions. Figure 2 shows that
the Gorreana (G) tea had a greater capacity to inhibit lipid peroxidation (AACG = 746.7).
However, there were no significant differences (p > 0.05) among the Gorreana (G), Chá
Camélia (AACCA = 600), and Happy Flora (AACH = 650) teas.
Additionally, considering the AAC of teas produced from fresh leaves (G, H, CA),
C. sinensis flower infusions, (CA), and teas/herbal infusions obtained from their reuse
(*2,*3,*4), it appears that the green tea obtained from the leaves of the Chá Camélia tea plan-
tation showed the highest capacity to retain its antioxidant activity (85%) compared with
the other samples (Gorreana ≈68.3%; Chá Camélia (flowers) ≈5.8%; Happy Flora ≈60.5%).
As far as we know, the β-carotene bleaching assay has not been used before to evaluate the
capacity of green tea infusions to inhibit lipid peroxidation, and therefore, it is not possible
to compare our results to those of other research studies.
600
AA
400
200
Foods 2022, 11, 1158 0 9 of 23

G G2 G3 G4 H H2 H3 H3 C CA2 CA3 CA4 CB CB2 CB3 CB4
Samples
β-carotene bleaching inhibition test

1000
800
600
AAC 400
200
0
G G2 G3 G4 H H2 H3 H4 CA CA2 CA3 CA4 CB CB2 CB3 CB4
Samples
Figure 2. Comparison of the antioxidant activity of different green teas and C. sinensis flower infusions.
Figure 2
Results of β-carotene bleaching inhibition test are expressed as antioxidant activity coefficients (AAC).
G = Gorreana; H = Happy Flora; CA = Chá Camélia (leaves); CB = Chá Camélia (flowers). The
numbers 2, 3, and 4 stand for the 2nd, 3rd and 4th infusions obtained by reusing the green tea leaves
(G, H, CA) or C. sinensis flowers (CB).
Total phenolic compounds

500 Total phenolic compounds
500
400
teatea
400
GAE/ml
300
GAE/ml
300
200
mgmg
200
100
100
0
0 G G2 G3 G4 H H2 H3 H4 CA CA2 CA3 CA4 CB CB2 CB3 CB4
Samples
Samples
Figure Comparison
Figure3. 3. Comparison of the
of total
the phenolic compounds
total phenolic of different
compounds of green and C. teas
teasgreen
different sinensis
andflower
C.
Figure
sinensis3.flower
infusions. Comparison
Resultsinfusions.ofResults
of TPC arethe total
expressed phenolic
of TPC
in mg of compounds
are expressed
gallic acid in mgofofdifferent
gallic acidgreen
equivalent per g of tea teas per
equivalent
(mg andgC.
GAE/g of
tea).
sinensis
Gtea flower infusions.
(mg GAE/gHtea).
= Gorreana; Results
G = Gorreana;
= Happy of TPC
Flora; CAH= =Chá are expressed
Happy in mg
Flora;(leaves);
Camélia CA = Chá of gallic
CB Caméliaacid equivalent
(leaves);
= Chá Camélia per g
CB = Chá
(flowers). of
The
tea (mg GAE/g
Camélia
numbers (flowers).
2, 3, andtea).
4 TheG numbers
stand = for
Gorreana;
2, 3,H
the 2nd, = and
and
3rd Happy
4th Flora;
4 stand for the
infusionsCA2nd,= Chá
3rd Camélia
obtained and (leaves);
4th infusions
by reusing CB tea
= Chá
obtained
the green by
leaves
Camélia
reusing (flowers).
the green Theleaves
tea numbers (G,2,
H,3,CA)
andor4C.
stand for the
sinensis 2nd, 3rd
flowers and 4th infusions obtained by
(CB).
reusing the green tea leaves (G, H, CA) or C. sinensis flowers (CB).
Total flavonoids compounds
150
Total flavonoids compounds
150
teatea
100
ml ml
100
ECE/
ECE/
50
mgmg
50
0
0 G G2 G3 G4 H H2 H3 H4 CA CA2 CA3 CA4 CB CB2 CB3 CB4
Samples
Samples
Figure 4. Comparison of the total flavonoid compounds (TFC) of different green teas and C.
Figure
Figure
sinensis Comparison
4.4.flowers’
Comparison ofofthe
infusion. thetotal flavonoid
total
Results offlavonoid
TFC are compounds
expressed(TFC)
compounds mgofofdifferent
as(TFC) green
ofepicatechin
different green and C.
teas teas
equivalent sinensis
and
per gC.
of
sinensis
tea (mg ECE/ml tea). G = Gorreana; H = Happy Flora; CA = Chá Camélia (leaves); CB = gChá
flowers’ flowers’
infusion.infusion.
Results Results
of TFC of
are TFC are expressed
expressed as mg as
of mg of epicatechin
epicatechin equivalent
equivalent per g per
of tea of
(mg
tea (mg ECE/ml
Camélia
ECE/ml G tea).
(flowers).
tea). TheGnumbers
= Gorreana;
= Gorreana; 3,Hand
H =2,Happy = Happy
4 standFlora;
Flora; for =the
CA CA
Chá =Camélia
2nd, Chá Camélia
3rd and (leaves);
4th infusions
(leaves); CB =Camélia
CB = obtained
Chá Cháby
Camélia
(flowers).(flowers).
reusing theThegreen Theleaves
tea
numbers numbers (G,2,
2, 3, and 4H, 3,CA)
standandfor
or4C.stand
the for3rd
sinensis
2nd, the 2nd,4th
flowers
and 3rd
(CB).and 4th infusions
infusions obtained byobtained
reusing bythe
reusing
green teathe green
leaves (G,tea
H,leaves
CA) or(G, H, CA) or
C. sinensis C. sinensis
flowers (CB). flowers (CB).
Foods 2022, 11, 1158 10 of 23
DPPH radical inhibition test

200
TEAC (μg TE/ml tea)

150
100
50
0
Samples
Figure 5. Comparison of the antioxidant activity of different green teas and C. sinensis flower infusions.
Results of DPPH radical inhibition test are expressed in µg Trolox equivalent per g of tea (µg TE/mL
tea). G = Gorreana; H = Happy Flora; CA = Chá Camélia (leaves); CB = Chá Camélia (flowers). The
numbers 2, 3, and 4 stand for the 2nd, 3rd and 4th infusions obtained by reusing the green tea leaves
Analysis of Figure 5 shows that the green tea obtained from the leaves of the Chá
Camélia tea plantation had the highest capacity to capture DPPH• free radicals
(≈139.8 µg TE/mL). However, excluding tea/herbal infusions produced through the third
reuse of leaves/flowers, there were no significant differences (p < 0.05) among tea varieties
regarding antioxidant capacity. The infusion of Chá Camélia green tea obtained from leaves
had the highest potential to retain antioxidant capacity (≈97.3%) when compared with the
other infusions obtained from flowers (≈73%) or leaves Gorreana (≈62.4%) and Happy
Flora (≈78.8%).
Several studies have used the DPPH radical scavenging assay to evaluate the antioxi-
dant capacity of green tea infusions. However, past publications reported the results of the
DPPH assay in different units, so we were unable to compare those results with those of
the current study.
Analysis of Figure 3 indicates significant superiority (p < 0.05) of the Portuguese
tea samples obtained from leaves in terms of total phenolic compound content, with
the Gorreana being considerably (p < 0.05) richer in phenolic compounds (≈428.4 mg
GAE/mL tea) than the Chá Camélia (≈385.5 mg GAE/mL tea). Considering the first and
fourth infusions, the Chá Camélia tea showed the highest potential to retain phenolic
compounds (≈37.4%). However, the difference was not as discrepant as for the Gorreana
tea (≈30%). It should be noted that, despite presenting the second-highest potential
for retention of phenolic compounds (≈34.5%), the infusion obtained from flowers of
C. sinensis (Chá Camélia), presented much lower values (≈144.9 mg GAE/mL tea) than
the two aforementioned green teas. In addition, is important to note that even though
the Happy Flora tea had the lowest values, it had the highest capacity to retain phenolic
compounds (≈93.6) when considering the first and second infusions.
A review of the literature allowed us to verify that, even though TPC is a widely
used parameter for evaluating the antioxidant activity of tea infusions, it is challenging to
compare TPC values with those obtained in other studies. Even though the Folin–Ciocâlteu
reagent method is one of the main techniques used to determine TPC, many procedures
have been applied. For instance, Kodama et al. [48] and Almeida et al. [49] determined the
TPC of tea infusions using the Folin–Ciocâlteu reagent method but according to Singleton,
Orthofer, and Lamuela-Raventos (1998) [50]. Komes et al. (2010) [51] used the same reagent
but determined the TPC according to the modified method of Lachman, Hosnedl, Pivec,
and Orsák (1998) [52]. In addition, the units in which the results have been displayed have
varied among studies. For instance, Kodama et al., 2010 expressed the results as mg of
catechin equivalent/200 mL.
Foods 2022, 11, 1158 11 of 23
Finally, Figure 4 shows values similar to those in Figure 3, which was expected. Thus,
Portuguese teas obtained from the leaves had higher total flavonoid content. However,
although the Gorreana tea was richer in flavonoids (≈130 ECE/mL tea), there were no
significant differences (p > 0.05) from the Chá Camélia tea (≈113.3 mg ECE/mL tea). Once
again, the Chá Camélia tea was superior in retention potential (44.9%) to other samples.
It should be noted that although Happy Flora had a higher retention potential than the
Gorreana tea (approximately 42.8% and 35%, respectively), the total flavonoid content in
Happy Flora tea (68 mg ECE/mL tea) was much lower than that in the Gorreana tea. Our
infusions presented higher total flavonoid content than the matcha green teas evaluated by
Jakubczyk et al. [53]. In addition, the TPC and TFC values of our infusions were higher
than those of the infusions evaluated by Komes et al. (2010) [51], among which the green
tea blend Rose of the Orient had lower TPC and TFC values (880 and 440 mg/L, GAE,
respectively), whereas the bagged Twinings of London green tea (2560 and 1920 mg/L
GAE, respectively) and powdered matcha green tea (2230 and 1630 mg/L, respectively)
had higher TPC and TFC values
3.1.2. Evaluation of the Antioxidant Capacity of Green Tea Extracts

In the present study, solid–liquid extractions and Soxhlet extractions were performed
simultaneously using the teas previously evaluated to compare the antioxidant capacity
and yield of green tea extracts. Chá Camélia tea varieties were excluded from this trial
because of the limited number of samples available.
In a brief analysis of Figures 6–9, it is possible to conclude that the extracts obtained
through the method described by Andrade et al. [36] demonstrated superior antioxidant
capacity to that of the extracts obtained through solid–liquid extraction with the Soxhlet
apparatus. In a more detailed analysis, Figure 6 confirms that the extracts obtained by the
conventional solid–liquid extraction method inhibited lipid peroxidation more efficiently,
presenting AAC values around 1500. However, there were no substantial differences among
the analyzed green tea samples when the same extraction method was applied (p > 0.05).
Furthermore, Figure 7 demonstrates that the extract of Gorreana obtained by conventional
solid–liquid extraction contained a higher content on total flavonoids (701.5 mg ECE/g extract).
However, the results shown in Figure 8 do not support the previously established premise.
Foods 2022, 11, x FOR PEER REVIEW 14 of 26
By analyzing Figure 8, it can be inferred that the extracts obtained through Soxhlet extraction
have a greater capacity to capture free radicals DPPH• (Gorreana ≈ 138.2 µg TE/g extract;
Happy Flora ≈ 131.2 µg TE/g extract) than extracts obtained through the conventional solid–
among the analyzed
liquid extraction green
method (Gorreana ≈ 118
tea samples when the same
µg E.T./g/ Happy Flora ≈
extraction method
114 µgwas applied
E.T./g), with (p
no
>significant
0.05). differences among green tea extracts when applied the same extraction method
(p > 0.05).
Gorreana (Soxhlet)
✱✱✱✱
Gorreana (Conventional)
✱
✱✱✱✱
✱✱✱✱
Happy Flora (Soxhlet)

ns
✱✱✱✱
Happy Flora (Conventional)
0 500 1000 1500 2000

AAC
Figure 6. Comparison of green tea extracts prepared by two different extraction methods. β-carotene
Figure 6. Comparison
bleaching inhibition testof greenaretea
results extractsasprepared
expressed AAC. ****by
= ptwo different
≤ 0.0001,* =p≤extraction
0.05, ns =methods.
p > 0.05.
β-carotene bleaching inhibition test results are expressed as AAC. **** = p ≤ 0.0001,* = p ≤ 0.05, ns = p
> 0.05.
Gorreana (Soxhlet)
✱✱✱✱
n
✱
0 AAC 1000
500 1500 2000
AAC
Figure 6. Comparison of green tea extracts prepared by two different extraction methods.
β-carotene
Figurebleaching inhibitionoftest
6. Comparison results
green teaare expressed
extracts as AAC.by****
prepared = p ≤different
two 0.0001,* =extraction
p ≤ 0.05, nsmethods.
=p
> 0.05.β-carotene bleaching inhibition test results are expressed as AAC. **** = p ≤ 0.0001,* = p ≤ 0.05, ns = p
Foods 2022, 11, 1158 12 of 23
> 0.05.
Gorreana (Soxhlet)
✱✱✱✱
Gorreana (Soxhlet)
✱✱✱✱
ns
✱✱✱✱
ns
ns
✱✱✱✱
✱✱✱✱
ns
✱✱✱✱
ns
ns
0 200 400 600 800
mg
0 ECE/g
200 extract
400 600 800
mg ECE/g extract
Figure 7. Comparison of green tea extracts prepared by two different extraction methods. Total
flavonoidFigure 7. Comparison
content is expressed of green tea extracts prepared by two different extraction methods. Total
Figure 7. Comparison of in mg of
green teaepicatechin equivalent
extracts prepared by per
twogdifferent
of extractextraction
(mg ECE/g extract). Total
methods.
flavonoid
****= pflavonoid content
≤ 0.0001, content
ns is expressed in mg of epicatechin equivalent per g of extract (mg ECE/g extract).
= p > is
0.05.
expressed in mg of epicatechin equivalent per g of extract (mg ECE/g extract).
**** = p ≤ 0.0001, ns = p > 0.05.
****= p ≤ 0.0001, ns = p > 0.05.
Gorreana (Soxhlet)
✱✱✱✱
Gorreana (Soxhlet)
✱✱✱✱
✱✱
✱✱✱✱
✱✱✱✱
✱✱
✱✱✱✱
✱✱✱✱
ns
✱✱✱✱
ns
✱✱✱✱
0 50 100 150
0µg TE/g50extract100 150
µgprepared
Foods 2022, 11, x FOR PEER REVIEW Figure 8. Comparison of green tea extracts TE/g extract 15 of 26of
by two different extraction methods. Results
Figure 8. Comparison of green tea extracts prepared by two different extraction methods. Results
DPPH radical inhibition test are expressed as mg Trolox equivalent per g of extract (µg TE/g extract).
of DPPH
Figure radical inhibition oftest are expressed as mg Trolox equivalent per g of extract (µg TE/g
**** = p8.≤Comparison
0.0001, ** = p ≤green tea=extracts
0.01, ns p > 0.05.prepared by two different extraction methods. Results
extract). **** = p ≤ 0.0001, ** = p ≤ 0.01, ns = p > 0.05.
of DPPH radical inhibition test are expressed as mg Trolox equivalent per g of extract (µg TE/g
extract). **** = p ≤ 0.0001, ** = p ≤ 0.01, ns = p > 0.05.
Gorreana (Soxhlet)
ns
ns
ns
ns
✱✱

✱
0 200 400 600 800 1000

mg GAE/g extract
phenolic compound content is expressed as mg of gallic acid equivalent per g of extract (mg GAE/g
phenolic compound content is expressed as mg of gallic acid equivalent per g of extract (mg GAE/g
extract). ** = p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.
extract). ** = p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.
Furthermore, regarding total phenolic compound content, there were no significant
Furthermore, Figure 7 demonstrates that the extract of Gorreana obtained by con-
differences (p > 0.05) between Gorreana extracts, even when different extraction methods
ventional solid–liquid
were applied. extraction
However, as showncontained a higher
in Figure content differences
9, significant on total flavonoids (701.5
(p < 0.05) mg
between
ECE/g extract). However, the results shown in Figure 8 do not support the previously
the Happy Flora extracts were observed. Furthermore, there were considerable differences
established
between the premise.
Happy By analyzing
Flora Figure 8, through
extract obtained it can beconventional
inferred thatsolid–liquid
the extracts extraction
obtained
through Soxhlet extraction have a greater capacity to capture free radicals
(≈758.3 mg GAE/g extract) and the Gorreana extract obtained through the same methodDPPH● (Gor-
reana ≈ 138.2 µg TE/g extract; Happy Flora ≈ 131.2 µg TE/g extract) than extracts obtained
through the conventional solid–liquid extraction method (Gorreana ≈ 118 µg E.T./g/
Happy Flora ≈ 114 µg E.T./g), with no significant differences among green tea extracts
when applied the same extraction method (p > 0.05).
Furthermore, regarding total phenolic compound content, there were no significant
Foods 2022, 11, 1158 13 of 23
(≈863.1 mg GAE/g extract). In addition, there were considerable differences between the
previously mentioned Happy Flora extract and the Gorreana extract obtained by Soxhlet
extraction (≈833.7 mg GAE/g extract).
Overall, the antioxidant capacity of our extracts was higher than that of the extracts
from a study by Martins et al. (2018) [54]. All of our extracts presented higher AAC
except Gorreana extracts obtained through Soxhlet extraction. According to our results,
the Happy Flora extract (conventional solid–liquid extraction) exhibited an AAC of 1536,
the Gorreana extract exhibited an AAC of 1488, and the Happy Flora extract (Soxhlet)
exhibited an AAC of 1210, while according to the results of Martins et al., extracts from
commercial green tea, green tea from capsules, “Hysson” green tea, and “Encosta da Bruma”
green tea exhibited AACs of 918, 1079, 1144, and 1178, respectively. Only the Gorreana
extracts obtained through Soxhlet extraction exhibited a lower, but still high, AAC (1051)
than the green tea extracts from the previously mentioned study. In addition, our DPPH
results were very high. Extracts from Gorreana (conventional), Gorreana (Soxhlet), Happy
Flora (conventional), and Happy Flora (Soxhlet) exhibited Trolox equivalent antioxidant
capacities (TEAC values expressed in µg TE/g extract) of 117.9, 138.2, 114, and 131.2,
respectively. According to Martins et al., extracts from commercial green tea, green tea
from capsules, “Hysson” green tea, and “Encosta da Bruma” green tea exhibited TEACs
(mg TE/g extract) of 0.917 ± 0.09, 0.560 ± 0.02, 0.602 ± 0.12, and 0.693 ± 0.07, respectively.
Furthermore, our extracts were richer in total polyphenols and total flavonoids than
those evaluated by Martins et al. (2018) [54]. As previously mentioned, extracts from
Gorreana (conventional), Gorreana (Soxhlet), Happy Flora (conventional), and Happy Flora
(Soxhlet) exhibited TPCs of 863.1, 833.7, 758.3, and 845.3 mg GAE/g extract, respectively,
while extracts from commercial green tea, green tea from capsules, “Hysson” green tea,
and “Encosta da Bruma” green tea exhibited TPCs of 416 ± 9.95, 272 ± 2.34, 330 ± 4.68,
and 361 ± 3.22 mg GAE/g extract, respectively.
Regarding the flavonoid content, extracts from Gorreana (conventional), Gorreana
(Soxhlet), Happy Flora (conventional), and Happy Flora (Soxhlet) exhibited TFCs of 701.5,
547.6, 500.2, and 500.0 mg ECE/g extract, respectively, while extracts from commercial
green tea, green tea from capsules, “Hysson” green tea, and “Encosta da Bruma” green
tea exhibited TFCs of 148 ± 0.2, 139 ± 9.59, 184 ± 0.64, and 165 ± 7.03 mg ECE/g extract,
respectively.
Furthermore, compared with the extract from Castro et al. (2019) [17], our extracts’
values were much higher, as their extract presented an AAC of 379.24 ± 10.43 mg GAE/g
of extract and a TFC of 119.81 ± 11.70 mg ECE/g of extract.
Finally, after a global analysis of the previously mentioned tests, Gorreana green tea
extract was chosen to be incorporated in the whey protein film. Theoretically, the Chá
Camélia extract (leaves) would also be an excellent option because of the tea’s high retention
potential and sensory characteristics. Additionally, solid–liquid extraction with the Soxhlet
apparatus was selected as the extraction method, since it presented a higher yield (43%)
than conventional solid–liquid extraction (5.6%).
3.2. Antioxidant and Antimicrobial Properties of the Active Film

After the films were developed, their antioxidant properties and antimicrobial proper-
ties were evaluated. The films obtained showed good mechanical properties. The active
films were characterized by an olive-green hue and a light green tea aroma. Additionally,
there was no solubilization between the films and the fresh cheeses.
3.2.1. Evaluation of the Antioxidant Capacity of the Whey Protein-Based Films

After producing the active films, migration tests were carried out, and their antioxidant
capacity was evaluated.
As expected, the active film incorporated with 2.5% green tea extract from the Gorreana
plantation exhibited a higher antioxidant capacity than the blank film (no added antioxidant
extract). As shown in Figures 10 and 11, the film incorporated with tea had not only a
tionally, there was no solubilization between the films and the fresh cheeses.
3.2.1. Evaluation of the Antioxidant Capacity of the Whey Protein-Based Films

After producing the active films, migration tests were carried out, and their antiox-
idant capacity was evaluated.
Foods 2022, 11, 1158 14 of 23
As expected, the active film incorporated with 2.5% green tea extract from the Gorreana
plantation exhibited a higher antioxidant capacity than the blank film (no added antioxidant
extract). As shown in Figures 10 and 11, the film incorporated with tea had not only a high
high capacity
capacity to inhibit
to inhibit lipid peroxidation
lipid peroxidation (≈595.4
(≈595.4 AAC) AAC)
but butcapacity
a high a high capacity
to capturetoDPPH●
capture
DPPH • free radicals ( ≈ 121 µg TE/g). However, compared with the results
free radicals (≈121 µg TE/g). However, compared with the results obtained in the evaluationobtained
ofinthe
theextract
evaluation of the obtained
of Gorreana extract ofbyGorreana obtained by
Soxhlet extraction, Soxhlet
there extraction,ofthere
were reductions 43% were
and
11% in the results for the β-carotene bleaching and DPPH radical tests, respectively. radical
reductions of 43% and 11% in the results for the β-carotene bleaching and DPPH
tests, respectively.
Camellia sinensis extract
✱✱✱✱
Blank

0 200 400 600 800
AAC
Figure
Figure10.
10.Results
Resultsofofthe
theantioxidant
antioxidantcapacity
capacityofofa aβ-carotene
β-carotenebleaching
bleachinginhibition
inhibitiontest teston
onthe
theactive
active
films after a migration test at 40 °C
◦ for 10 days (active film—C. sinensis extract vs. control—blank
films after a migration test at 40 C for 10 days (active film—C. sinensis extract vs. control—blank
film).
film).β-carotene
β-carotenebleaching
bleachinginhibition
inhibitiontest
testresults
resultsare
areexpressed
expressedasasAAC.
AAC.****
****==p p≤ ≤
0.0001.
0.0001.
Blank
0 50 100 150
TEAC (µg TE/g)
Figure 11. Results of the antioxidant capacity of the active films after a migration test at 40 ◦ C for 10
Figure 11. Results of the antioxidant capacity of the active films after a migration test at 40 °C for 10
days(active
days (activefilm—C. sinensis extract
film—C. sinensis extract vs.
vs. control—blank
control—blankfilm).
film).DPPH
DPPHradical
radicalinhibition test
inhibition results
test are
results
expressed in mg Trolox equivalent per g of film (µg TE/g film). * = p ≤ 0.05.
are expressed in mg Trolox equivalent per g of film (µg TE/g film). * = p ≤ 0.05.
After a migration test carried out using ethanol 95% (v/v) as a food simulant (simu-
lant of fatty foods), the new active film presented a TPC of 121.3 mg GAE/g and a TFC
lant of fatty foods), the new active film presented a TPC of 121.3 mg GAE/g and a TFC of
of 55.4 mg ECE/g (Figures 12 and 13). However, compared with the extract of Gorre-
55.4 mg ECE/g (Figures 12 and 13). However, compared with the extract of Gorreana
ana obtained by Soxhlet extraction, there were decreases of 93.3% and 76% in TPC and
obtained by Soxhlet extraction, there were decreases of 93.3% and 76% in TPC and TFC,
TFC, respectively.
respectively.
Besides films prepared with green tea extract, there were films prepared with green
tea. For these films, the values obtained in the β-carotene bleaching and DPPH radical tests
were very similar to those obtained during the evaluation of the antioxidant capacity of
Gorreana green tea. However, there were reductions of 71.7% and 57.4% in the content
Camellia
of sinensis
phenolic extract and flavonoids, respectively. However, it should be noted that
compounds
the active films developed from the incorporation of green tea did not demonstrate any
✱
antioxidant capacity.
Blank
0 50 100 150 200

mg GAE/g
Figure 12. Results of the total phenolic content of the active films after a migration test at 40 °C for
are expressed in mg Trolox equivalent per g of film (µg TE/g film). * = p ≤ 0.05.
lant of fatty foods), the new active film presented a TPC of 121.3 mg GAE/g and a TFC of
55.4 mg ECE/g (Figures 12 and 13). However, compared with the extract of Gorreana
Foods 2022, 11, 1158
obtained by Soxhlet extraction, there were decreases of 93.3% and 76% in TPC and 15 of 23
TFC,
respectively.
✱
Blank
0 50 100 150 200

mg GAE/g
◦ for 10
Foods 2022, 11, x FOR PEER REVIEW Figure 12. Results of the total phenolic content of the active films after a migration test at 40 18C of 26
Figure 12. Results of the total phenolic content of the active films after a migration test at 40 °C for
days (active film—C. sinensis extract vs. control—blank film), expressed in mg gallic acid equivalent
10 days (active film—C. sinensis extract vs. control—blank film), expressed in mg gallic acid
per g of film
equivalent per(mg
g ofGAE/g
film (mgfilm). p ≤ 0.05.
* = film).
GAE/g * = p ≤ 0.05.
✱✱
Blank
0 20 40 60 80
mg ECE/g
Figure 13. Results of the total flavonoid content of the active films after a migration test at 40 ◦ C
Figure 13. Results of the total flavonoid content of the active films after a migration test at 40 °C for
10fordays
10 days (active
(active film—C.
film—C. sinensis
sinensis extract
extract vs.vs. control—blank
control—blank film),
film), expressedininmg
expressed mgepicatechin
epicatechin
equivalent per g of film (mg ECE/g). ** = p ≤
equivalent per g of film (mg ECE/g). ** = p ≤ 0.01. 0.01.
According to the literature, very few studies have evaluated the antioxidant capacity
Besides films prepared with green tea extract, there were films prepared with green
of whey protein-based films incorporated with green tea extracts. Pluta-Kubica et al.
tea. For these films, the values obtained in the β-carotene bleaching and DPPH radical
(2021) [16] and Chalob and Abdul-Rahman (2018) [55] developed edible films using whey
tests were very similar to those obtained during the evaluation of the antioxidant capac-
protein isolates as biopolymeric matrices and green tea extracts as antioxidant agents.
ity of Gorreana green tea. However, there were reductions of 71.7% and 57.4% in the
However, the first study used both Furcellaran and whey protein isolate as a biopolymeric
content of phenolic compounds and flavonoids, respectively. However, it should be
matrix, and therefore, it was not possible to compare accurately the antioxidant capacity
noted that the active films developed from the incorporation of green tea did not
obtained therein with that obtained here, since we used only whey protein concentrate
demonstrate any antioxidant capacity.
as a biopolymeric matrix. The second study developed a whey protein isolate-based film
According to the literature, very few studies have evaluated the antioxidant capacity
and evaluated its antioxidant capacity using a procedure in which the film was cut into
of whey protein-based films incorporated with green tea extracts. Pluta-Kubica et al.
aliquots, dissolved in deionized water, and centrifuged. A portion of the solution was
(2021)
mixed[16] and
with a Chalob
methanolic and Abdul-Rahman
solution of DPPH, (2018) [55] developed
vortexed, and thenedible films using
incubated. whey
Finally, the
protein isolates as biopolymeric matrices and green tea extracts as antioxidant
solution was centrifuged, and the absorbance was measured. Unfortunately, this procedure agents.
However, the from
was different first study used
that used bothassay
in this Furcellaran andthe
to evaluate whey protein capacity
antioxidant isolate asand
a biopoly-
therefore
meric matrix, and therefore,
does not allow comparison. it was not possible to compare accurately the antioxidant
capacity obtained ittherein
In addition, with that
is important obtained
to remark thathere, since we
both studies used
used wheyonlyprotein
whey isolate
proteinin
concentrate as a biopolymeric matrix. The second study developed a whey
the formulation of the edible film, and in this study, we used whey protein concentrate, protein iso-
late-based
which may influence the film’s antioxidant capacity. As far as we know, according tothe
film and evaluated its antioxidant capacity using a procedure in which the
film was cut
available into aliquots,
bibliography, nodissolved
studies havein deionized water,the
fully compared andantioxidant
centrifuged. A portion
activity of the
of WPI and
solution
WPC films.was mixed with a methanolic solution of DPPH, vortexed, and then incubated.
Finally, the solution
Moreover, was centrifuged,
this research assessed and the absorbance
antioxidant capacity wasonlymeasured.
through DPPH Unfortunately,
scavenging
this procedure was different from that used in this assay to evaluate the
assay, and this parameter does not provide meaningful information about the film’s actual antioxidant ca-
pacity and therefore does not allow comparison.
antioxidant capacity when considered alone [33].
In addition, it is important to remark that both studies used whey protein isolate in
the formulation of the edible film, and in this study, we used whey protein concentrate,
which may influence the film’s antioxidant capacity. As far as we know, according to the
available bibliography, no studies have fully compared the antioxidant activity of WPI
and WPC films.
Foods 2022, 11, 1158 16 of 23
3.2.2. Evaluation of the Lipid Oxidation Status of Packaged Goat and Mixture
Fresh Cheeses
The TBARS (thiobarbituric acid reactive substances) assay detects lipid peroxidation
through measuring malondialdehyde (CH2 (CHO)2 ) (MDA) [17]. For this assay, a calibration
curve (y = 17.657x − 0.0326; r2 = 0.9905) was built by plotting different concentrations of
the fresh cheeses packaged with
1,1,3,3-tetramethoxypropane the µg/mL)
(10–55 whey protein-based
to express theactive film
results (incorporated
in mg with
of malonaldehyde
green tea extract) presented lower MDA content than those packaged
equivalent per kg of sample (mg MDA Eq/kg). Figure 14 demonstrates that all the fresh with the control
film (without
cheeses green tea
packaged withextract).
the whey Goatprotein-based
and mixture fresh
activecheeses packaged with
film (incorporated with thegreen
activetea
film exhibited 3.2 and 3.7 mg MDA/kg sample, respectively, while those
extract) presented lower MDA content than those packaged with the control film (without packaged with
thegreen
control film exhibited
tea extract). Goat and 4.2mixture
and 4.4fresh
mg cheeses
MDA/kg sample,with
packaged respectively.
the active In
film addition,
exhibited
within
3.2 andthe3.7
same type of cheese,
mg MDA/kg sample, significant differences
respectively, while thosewerepackaged
found between
with the the cheeses
control film
wrapped
exhibited with
4.2 the
andactive
4.4 mgfilm and those
MDA/kg wrapped
sample, with the In
respectively. control film.within
addition, Furthermore,
the same
none
typeofofthe freshsignificant
cheese, cheese samples packaged
differences werewith
found the active and
between control wrapped
the cheeses films presented
with the
MDAactive film and those wrapped with the control film. Furthermore, none of the off
values above 0.5 mg MDA/kg sample, which is the threshold in which flavors
fresh cheese
aresamples
perceived [8]. However,
packaged with thein a previous
active analysis,
and control filmsafter 6 daysMDA
presented of storage,
valuesunpackaged
above 0.5 mg
mixture
MDA/kg fresh mixture
sample, cheese
which presented
is the threshold 5.2inmg MDA/kg
which sample.
off flavors These results
are perceived [8]. suggest
However,
that,
in awithin
previousa week, theseafter
analysis, active packages
6 days have the
of storage, potential to
unpackaged inhibitfresh
mixture freshmixture
cheese lipid
cheese
oxidation.
presentedNevertheless,
5.2 mg MDA/kg it is important
sample. Theseto remark
resultsthat TBARS
suggest has
that, somealimitations,
within week, thesesinceactive
it packages
depends on substances
have reactive
the potential to thiobarbituric
to inhibit fresh cheeseacid,lipid
which degrade Nevertheless,
oxidation. along with lipid it is
oxidation.
important to remark that TBARS has some limitations, since it depends on substances
reactive to thiobarbituric acid, which degrade along with lipid oxidation.
FMCB
✱✱
Samples
FMCA
FGCB
✱
FGCA
0 1 2 3 4 5
mg MDA Eq /kg
Figure 14. Results of TBARS assay for goat and mixture fresh cheese samples packaged with the
Figure 14. Results of TBARS assay for goat and mixture fresh cheese samples packaged with the
control film (without green tea extract) and active film (with green tea extract). FMCB—mixture fresh
control film (without green tea extract) and active film (with green tea extract). FMCB—mixture
cheese packaged with the control film; FMCA—mixture fresh cheese packaged with the active film;
fresh cheese packaged with the control film; FMCA—mixture fresh cheese packaged with the ac-
FGCB—goat
tive fresh cheese
film; FGCB—goat freshpackaged with the control
cheese packaged film;control
with the FGCA—goat fresh cheese fresh
film; FGCA—goat packaged with
cheese
the active film. * = p ≤ 0.05, ** = p ≤ 0.01.
packaged with the active film. * = p ≤ 0.05, ** = p ≤ 0.01.
Andrade
Andrade et et
al.al.
[8][8] and
and Castro
Castro et et
al.al. [17],
[17], through
through thethe TBARS
TBARS assay,evaluated
assay, evaluated the
the
oxidation status of salami and smoked salmon, respectively, both packaged
oxidation status of salami and smoked salmon, respectively, both packaged with an ac- with an active
film similar to that used in this study. Andrade et al.’s results showed that after one
tive film similar to that used in this study. Andrade et al.’s results showed that after one
week, salami packaged with the active film presented approximately 12.5% less MDA
week, salami packaged with the active film presented approximately 12.5% less MDA
content when compared to the sample packaged with the control film. Castro et al. [17]
content when compared to the sample packaged with the control film. Castro et al. [17]
demonstrated that after one week, smoked salmon packaged with the active film had
demonstrated that after one week, smoked salmon packaged with the active film had
approximately 24% less MDA content than the control. Comparing the present study’s
approximately 24% less MDA content than the control. Comparing the present study’s
results with those of the previously mentioned studies, our active package had efficient
results with those of the previously mentioned studies, our active package had efficient
performance, as within one week, goat and mixture fresh cheese packaged with the active
performance, as within one week, goat and mixture fresh cheese packaged with the ac-
film had 24% and 15% less MDA content, respectively, than the corresponding fresh cheeses
tive film had 24% and 15% less MDA content, respectively, than the corresponding fresh
packaged with the control film. It is important to remark, however, that the food matrices
cheeses packaged with the control film. It is important to remark, however, that the food
were different and that fresh cheese is much more perishable than salami or salmon, which
matrices
explainswere different
the higher andofthat
levels fresh cheese
oxidation of freshischeese.
much more perishable than salami or
salmon, which explains the higher levels of oxidation of fresh cheese.
3.2.3. Microbiological Analysis of Packaged Fresh Cheeses

In this study, the total count of microorganisms at 30 °C, E. coli, and coagu-
lase-positive staphylococci were investigated, since these are indicators not only of food
Foods 2022, 11, 1158 17 of 23

3.2.3. Microbiological Analysis of Packaged Fresh Cheeses
In this study, the total count of microorganisms at 30 ◦ C, E. coli, and coagulase-positive
staphylococci were investigated, since these are indicators not only of food quality but of
production
complianceand withindistribution.
the scope Additionally,
of good hygiene since fresh cheeses
practices are usually
during cheese storedand
production at re-
dis-
frigerated temperatures (5 °C), a psychrophile count was carried out. The
tribution. Additionally, since fresh cheeses are usually stored at refrigerated temperatures counts of these
different
(5 ◦ C), a microorganisms
psychrophile count were
was carried
carriedout onThe
out. goat fresh of
counts cheese
these and mixture
different (sheep and
microorganisms
goat)
werefresh cheese
carried out onproduced oncheese
goat fresh the day. Furthermore,
and mixture (sheep microorganism
and goat) fresh counts
cheeseonproduced
the two
types
on theofday.
freshFurthermore,
cheeses wrapped with activecounts
microorganism films on
(active) andtypes
the two control filmscheeses
of fresh (without green
wrapped
tea extract—blank) were carried out after one week of storage at 5 °C.
with active films (active) and control films (without green tea extract—blank) were carried
out In terms
after oneofweekfoodofquality, 5 ◦ C.
storageitatcan be concluded from Figure 15 that the active films ef-
fectively inhibited the growth of mesophilic
In terms of food quality, it can be concluded microorganisms, and 15
from Figure thethat
goatthe
cheese coated
active films
with the active
effectively film presented
inhibited the growth a significantly
of mesophilic lower count of CFU/g
microorganisms, and(2.9
the ×goat
10 CFU/g)
6 than
cheese coated
the blank
with coatedfilm
the active cheese (1.1 × a10significantly
presented lower count of CFU/g (2.9 × 106 CFU/g)
7 CFU/g). In mixture fresh cheese (goat and sheep), than
the
colonies
the blank were countless;
coated however,
cheese (1.1 × 10 to7 the naked
CFU/g). eye, the
In mixture active
fresh film-coated
cheese (goat andsample
sheep),ap-the
peared
coloniesto were
have countless;
fewer colonies compared
however, to the eye,
to the naked blank thecoated
activesample (fresh
film-coated cheeseappeared
sample coated
with control
to have film).
fewer colonies compared to the blank coated sample (fresh cheese coated with
control film).
Goat Fresh Cheese
Goat Fresh Cheese (Blank)
Goat Fresh Cheese (Active)
Sheep and Goat Fresh Cheese
0 5×10 6 1×10 7 1.5×10 7

CFU/g
Figure 15. Results of total microorganism count at 30 ◦ C, expressed as colony-forming units per g of
Figure 15. Results of total microorganism count at 30 °C, expressed as colony-forming units per g of
freshcheese
fresh cheese (CFU/g
(CFU/g fresh
fresh cheese).
cheese). Total
Totalmicroorganism
microorganism counts
countswere carried
were out on
carried outgoat
on fresh
goat cheese
fresh
(Goat Fresh Cheese) and mixture fresh cheese (Sheep and Goat Fresh Cheese)
cheese (Goat Fresh Cheese) and mixture fresh cheese (Sheep and Goat Fresh Cheese) produced produced on the on
day.
Furthermore,
the both cheeses
day. Furthermore, were wrapped
both cheeses with the
were wrapped edible
with the active
ediblefilm (Goat
active filmFresh
(GoatCheese (Active)
Fresh Cheese
and Sheep
(Active) and and Goat
Sheep andFresh
GoatCheese (Active))
Fresh Cheese and with
(Active)) andthe control
with film (Goat
the control film Fresh
(Goat Cheese (Blank)
Fresh Cheese
(Blank) and Sheep
and Sheep andFresh
and Goat GoatCheese
Fresh Cheese (Blank))
(Blank)) and stored at 5 ◦ Cat
and stored in5order
°C in to
order to simulate
simulate standard
standard storage
storage conditions.
conditions. After
After one oneofweek
week of storage
storage at refrigerated
at refrigerated temperatures,
temperatures, a total microorganism
a total microorganism count was
count was
carried carried
out. out. Theofnumbers
The numbers colonies onof Goat
colonies
FreshonCheese
Goat Fresh
(Active)Cheese (Active)
and blank wereand blank were
uncountable and
uncountable and are therefore not shown.
are therefore not shown.
InInthe
thecontext
contextofoffood
food safety,
safety, considering that despite
considering that despite working
working as asaahygiene
hygieneindicator,
indica-
tor,
somesome strains
strains of E.
of E. colicoli
areare pathogenic,
pathogenic, Figures
Figures 1616 and
and 1717 show
show inhibitionofofthe
inhibition thegrowth
growthof
ofmicroorganisms
microorganisms inin the
the samples
samples coated
coated byby
thethe active
active film.
film. In In fact,
fact, in in goat
goat cheese
cheese (Figure
(Figure 16),
16), there was even a significant reduction in the number of colonies
there was even a significant reduction in the number of colonies (1.5 × 10 CFU/g) when (1.5 × 10 CFU/g)
when compared
compared with with
fresh fresh
cheese cheese produced
produced on theonday
the(1.8 102×CFU/g)
day×(1.8 102 CFU/g)
and,and, of course,
of course, with
with the blank
the blank coated
coated cheese
cheese (2.2 (2.2 2
× 10× 10 2 CFU/g).
CFU/g).
In the mixture cheese (Figure 17), there was no decrease in the number of colonies
when compared with fresh cheese produced on the day (1.6 × 104 CFU/g); however, the
cheese coated by the active film had a lower number of CFU/g (3.2 × 105 CFU/g) than
the blank-coated cheese (4.4 × 105 CFU/g). Note that neither coagulase + staphylococci nor
psychrophiles grew in any of the samples. At least six edible films have been developed to
date for unripened cheeses of which the antimicrobial activity has been assessed. Pluta-
Kubica et al. (2020) [14] developed active edible furcellaran/whey protein films with
yerba mate and white tea extracts and applied them to fresh soft rennet-curd cheese. The
Foods 2022, 11, 1158 18 of 23
microbiological quality of the cheese was determined through total count of bacteria (TBC),
yeast, molds, and coliforms. In 2021, Pluta-Kubica et al. [16] published another article
in which they applied furcellaran/whey protein isolate films containing extracts (Pu-erh
or green tea) to an acid-curd cheese. This study determined Lactococcus, TBC, yeast,
molds, and coliforms. Furthermore, Santacruz and Castro (2018) [28] developed a cassava
starch edible coating incorporated with Lactobacillus acidophilus and applied it to Manaba
fresh white cheese. The antibacterial activity of L. acidophilus was assessed against a
Salmonella strain previously isolated from Manaba cheese. The mesophilic aerobic bacteria
on the cheese were also determined. Moreover, Di Pierro et al. (2011) [15] developed
a chitosan/whey protein coating to extend ricotta cheese’s shelf life and determined its
antimicrobial activity against aerobic, mesophilic, and psychotropic microorganisms. In
addition, Mileriene et al. (2021) [13] evaluated the effect of liquid whey protein concentrate-
based edible coating enriched with cinnamon carbon dioxide extract on eastern European
curd cheese’s quality and shelf life. To assess the microbiological quality, they enumerated
TBC, lactic acid bacteria, Staphylococcus spp., Enterobacteria, coliforms, yeast, and molds.
Finally, Jutinico-Shubach et al. [29] evaluated the antilisterial activity of chitosan-based
edible coating incorporating cell-free supernatant from Pediococcus pentosaceus 147 on
the preservation of Colombian fresh cheese and performed microbiological analysis by
determination of L. monocytogenes, mesophilic bacteria, psychrophilic bacteria, and total
mold and yeast counts.
Most of the aforementioned research involved developing attractive edible films/coatings
to apply to unripened cheese surfaces. However, the microbiological quality of the various
unripened cheeses was evaluated through a wide variety of parameters, not allowing ad-
equate comparisons. In addition, it is essential to remark that the microbiological quality
varies between unripened cheeses, as some require starter cultures (preparations of living
Foods 2022, 11, x FOR PEER REVIEW microorganisms, which are deliberately added to foods to take advantage of the compounds or 21 of
products derived from their metabolism or enzymatic activity) to be produced [56]. Neverthe-
less, some authors have evaluated the microbiological quality of cheeses through enumeration
of mesophilic bacteria, allowing comparisons with the present study results.
Goat Fresh Cheese (Active)
Goat Fresh Cheese (Blank)
Goat Fresh Cheese
0 5×10 1 1×10 2 1.5×10 2 2×10 2 2.5×10 2

CFU/g
Figure 16. Results of the Escherichia coli colony count in goat fresh cheese, expressed as CFU per g
Figure
of fresh16. Results
cheese of the
(CFU/g Escherichia
fresh coliEscherichia
cheese). An colony count in goat
coli colony fresh
count wascheese, expressed
carried out as CFU per g
on goat fresh
fresh cheese (CFU/g fresh cheese). An Escherichia coli colony count was
cheese produced on the day (Goat Fresh Cheese). Furthermore, the cheese was wrapped with the carried out on goat fr
cheese produced
edible active on the
film (Goat day
Fresh (Goat(Active))
Cheese Fresh Cheese).
and with the Furthermore, the cheese
control film (Goat was wrapped
Fresh Cheese (Blank)) with
edible
and stored at 5 C in order to simulate standard storage conditions. After one week of storageFresh
active film
◦ (Goat Fresh Cheese (Active)) and with the control film (Goat at Che
(Blank)) and stored at 5 °C in order to simulate standard
refrigerated temperatures, an Escherichia coli colony count was carried out. storage conditions. After one week
storage at refrigerated temperatures, an Escherichia coli colony count was carried out.
Sheep and Goat Fresh Cheese (Active)

Figure 16. Results of the Escherichia coli colony count in goat fresh cheese, expressed as CFU per g of
fresh cheese (CFU/g fresh cheese). An Escherichia coli colony count was carried out on goat fresh
cheese produced on the day (Goat Fresh Cheese). Furthermore, the cheese was wrapped with the
edible active film (Goat Fresh Cheese (Active)) and with the control film (Goat Fresh Cheese
(Blank)) and stored at 5 °C in order to simulate standard storage conditions. After one week of
Foods 2022, 11, 1158 19 of 23
storage at refrigerated temperatures, an Escherichia coli colony count was carried out.
Sheep and Goat Fresh Cheese (Active)
Sheep and Goat Fresh Cheese (Blank)
Sheep and Goat Fresh Cheese
0 1×10 5 2×10 5 3×10 5 4×10 5 5×10 5

CFU/g
Figure 17. Results of the Escherichia coli colony count in mixture (sheep and goat) fresh cheese,
Figure 17. Results of the Escherichia coli colony count in mixture (sheep and goat) fresh cheese, ex-
expressed
pressed as CFU
as CFU perper g of
g of freshcheese
fresh cheese(CFU/g
(CFU/g fresh
fresh cheese).An
cheese). Escherichiacoli
AnEscherichia colicolony
colonycount
countwas
was
carried out on mixture fresh cheese produced on the day (Sheep and Goat Fresh
carried out on mixture fresh cheese produced on the day (Sheep and Goat Fresh Cheese). Fur- Cheese). Furthermore,
the cheesethe
thermore, was wrapped
cheese with the with
was wrapped ediblethe
active
ediblefilm (Sheep
active filmand Goatand
(Sheep Fresh
GoatCheese
Fresh (Active)) and
Cheese (Ac-
with and
tive)) the control
with thefilm (Sheep
control filmand Goatand
(Sheep Fresh Cheese
Goat Fresh(Blank))
Cheese and stored
(Blank)) and 5 ◦ C inatorder
atstored to order
5 °C in simulate
to
simulate standard storage conditions. After one week of storage at refrigerated temperatures,
standard storage conditions. After one week of storage at refrigerated temperatures, an Escherichia an
Escherichia
coli colonycoli colony
count wascount
carried was carried out.
out.
InInthe mixture
a study cheese (Figureet17),
by Pluta-Kubica there was
al. (2020) [14],no decrease
within in the the
one week, number
TBC ofof uncoated
colonies
when
fresh compared with fresh
soft rennet-curd cheesecheese produced
diminished on the
from dayCFU/g
8.8 log (1.6 × 10 to4 CFU/g); however,Inthe
8.6 log CFU/g. ad-
dition,coated
cheese after one week,
by the cheese
active film coated with furcellaran/whey
had a lower number of CFU/g (3.2 protein
× 10isolate
5 CFU/g) exhibited
than thea
TBC of 8.8 logcheese
blank-coated CFU/g, (4.4cheese
× 105coated
CFU/g). with furcellaran/whey
Note protein isolate
that neither coagulase incorporated
+ staphylococci nor
with yerba mate
psychrophiles grewextract
in any exhibited a TBCAt
of the samples. of 8.6 log
least sixCFU/g, and cheese
edible films coated
have been with furcel-
developed to
laran/whey
date proteincheeses
for unripened isolate incorporated
of which thewith white tea extracts
antimicrobial activity exhibited
has been aassessed.
TBC of 8.5 log
Plu-
CFU/g. Even
ta-Kubica et al.though differences
(2020) [14] developed between
activethe activefurcellaran/whey
edible films and the control proteinwere minimal
films with
or nonexistent, the authors were able to obtain good results in the scope
yerba mate and white tea extracts and applied them to fresh soft rennet-curd cheese. The of their research
because TBC, in quality
microbiological fresh softofrennet-curd
the cheese cheese, includes primarily
was determined throughlactictotalacid bacteria,
count which
of bacteria
are notyeast,
(TBC), spoilage microorganisms.
molds, and coliforms. In 2021, Pluta-Kubica
In 2021, Pluta-Kubicaet al.et[16]
al.demonstrated
[16] published that within
another
one week,
article the TBC
in which theyofapplied
uncoated fresh acid-curd cheese
furcellaran/whey proteindiminished
isolate films from 7.5 log CFU/g
containing extractsto
6.5 log CFU/g.
(Pu-erh or greenIntea)
addition, after one week,
to an acid-curd cheese.cheese coateddetermined
This study with furcellaran/whey
Lactococcus,protein
TBC,
isolate exhibited a TBC of 5.9 log CFU/g, cheese coated with furcellaran/whey
yeast, molds, and coliforms. Furthermore, Santacruz and Castro (2018) [28] developed protein
a
isolate incorporated with green tea extract exhibited a TBC of 5.9 log CFU/g, and cheese
coated with furcellaran/whey protein isolate incorporated with Pu-erh extracts exhibited a
TBC of 6.8 log CFU/g. In this study, however, none of the edible films used improved the
microbiological quality of an acid-curd cheese.
Santacruz and Castro (2018) [26] demonstrated that within ten days, mesophilic
bacteria on uncoated Manaba cheese increased from 8.60 log CFU/g to 9.20 log CFU/g.
After ten days, Manaba cheese coated with cassava starch film containing free L. acidophilus
presented 4.10 log CFU/g, while cassava starch film containing encapsulated L. acidophilus
presented 5.90 log CFU/g. Moreover, according to Di Pierro et al. (2011) [15], the viable
numbers of mesophilic bacteria on uncoated ricotta cheese after one week increased from
≈6.5 log CFU/g to ≈7.0 log CFU/g, while on the coated ricotta cheese, the viable numbers
of mesophilic bacteria were maintained (≈6.5 log CFU/g). Finally, in a 2021 study by
Mileriene et al. [13], the TBC diminished from 7 to 4 log CFU/g in all samples during
storage (31 days). However, after ten days of storage, the curd cheese TBC increased
from 6.91 log CFU/g to 7.15 log CFU/g, and the coated curd cheese TBC increased from
6.92 log CFU/g to 7.26 log CFU/g. On the other hand, vacuum-packed curd cheese’s
and coated and vacuum-packed curd cheese’s TBCs diminished from 6.91 log CFU/g to
6.39 log CFU/g and from 6.90 log CFU/g to 6.47 log CFU/g, respectively.
Regarding the viable numbers of mesophilic bacteria, converting CFU/g to log CFU/g,
after one week, our uncoated goat cheese presented ≈7.0 log CFU/g, and our coated goat
cheese presented ≈6.5 log CFU/g. In addition, it is important to recall that Latin-style fresh
Foods 2022, 11, 1158 20 of 23
cheese manufacture does not require starter cultures. This actively demonstrates that our
whey protein-based edible film incorporated with green tea extract had good performance
compared with the formerly developed ones. However, this parameter alone does not
allow any conclusions.
4. Conclusions
In the present study, a screening of the antioxidant capacity of Portuguese and Asian
green tea infusions was successfully performed. The green tea provided by Chá Camélia
was characterized for the first time, as was the infusion prepared with flowers of C. sinensis.
The former revealed a great antioxidant capacity and outstanding potential to retain an-
tioxidant capacity. Moreover, extracts from two brands (a Portuguese and an Asian brand)
of green tea were prepared, and their yields and antioxidant capacities were evaluated,
demonstrating their high antioxidant potential. It is important to remark that in future
studies, qualitative analysis of the extracts should be performed. Because of limitations in
the samples’ availability, Chá Camélia samples were not used to prepare extracts. However,
in the future, these samples should be considered. In addition, a comparison between
conventional solid–liquid extraction and Soxhlet extraction suggested that the former may
be the most adequate because of its yield. Furthermore, the use of the Soxhlet apparatus
does not allow stirring and may lead to the thermal degradation of compounds, since this
process exposes the samples to high temperatures for a long time [57].
Additionally, active whey protein-based films incorporated with green tea extracts
(2.5%) were successfully developed, and their application as a packaging material for
Latin-style fresh cheeses effectively preserved this product.
Evaluation of the antioxidant capacity of the developed films was also carried out
and indicated that whey protein-based films incorporated with green tea extracts might
successfully prevent or inhibit lipid oxidation of fresh cheeses. In addition, through the
TBARS assay, the capacity of the new active film to delay lipid oxidation of fresh cheese
during storage was confirmed.
Tested over a week, the cheese samples wrapped with active edible films were charac-
terized by lower counts of mesophilic bacteria and E. coli. However, the active edible films
altered the color and hardness of fresh cheeses. Further studies are required to fully assess
whether this package can extend Latin-style fresh cheese’s shelf life and to evaluate the
impact of the active films developed on the organoleptic quality of Latin-style fresh cheese
samples. In future studies, the total microorganism count at 30 ◦ C should be repeated
for mixture fresh cheese wrapped with control and active films in order to obtain more
results. Additional studies should be performed to fully evaluate the antimicrobial capacity
concerning foodborne bacteria such as Listeria monocytogenes.
The developed whey protein-based films incorporated with green tea extracts are
disruptive packaging materials that have been successfully used to preserve Latin-style
fresh cheese. Furthermore, this packaging is environmentally friendly, as it is biodegradable
and renewable and therefore generates zero waste. Moreover, its manufacture adds value
to whey, making it more profitable to companies. However, greater understanding of
the subject and sensory analysis are required to better understand the magnitude of the
cost/benefit ratio and whether or not this innovation would be accepted by consumers.
Author Contributions: Conceptualization, A.S.S.; data curation, J.R.; formal analysis, J.R. and M.L.;
funding acquisition, A.S.S. and F.R.; project administration, A.S.S. and F.R.; software, J.R.; supervision,
A.S.S. and F.R.; validation, M.L., O.C. and F.R.; visualization, J.R.; writing—original draft, J.R.;
writing—review and editing, O.C., A.S.S. and F.R. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was financially supported by the Faculty of Pharmacy of the University of
Coimbra (Master in Food Safety). The work was supported by UIDB/00211/2020 with funding from
FCT/MCTES through national funds.
Institutional Review Board Statement: Not applicable.
Foods 2022, 11, 1158 21 of 23
Informed Consent Statement: Not applicable.

Data Availability Statement: Data is contained within the article.
Acknowledgments: We acknowledge the samples kindly supplied by Chá Camélia and Queijaria Licínia.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Costa, M.J.; Maciel, L.C.; Teixeira, J.A.; Vicente, A.A.; Cerqueira, M.A. Use of Edible Films and Coatings in Cheese Preservation:
Opportunities and Challenges. Food Res. Int. 2018, 107, 84–92. [CrossRef] [PubMed]
2. Hinrichs, J. Incorporation of Whey Proteins in Cheese. Int. Dairy J. 2001, 11, 495–503. [CrossRef]
3. Coelho, M.C.; Silva, C.C.G.; Ribeiro, S.C.; Dapkevicius, M.L.N.E.; Rosa, H.J.D. Control of Listeria Monocytogenes in Fresh Cheese
Using Protective Lactic Acid Bacteria. Int. J. Food Microbiol. 2014, 191, 53–59. [CrossRef] [PubMed]
4. Brandelli, A.; Daroit, D.J.; Corrêa, A.P.F. Whey as a Source of Peptides with Remarkable Biological Activities. Food Res. Int. 2015,
73, 149–161. [CrossRef]
5. Asensio-Grau, A.; Peinado, I.; Heredia, A.; Andrés, A. In Vitro Study of Cheese Digestion: Effect of Type of Cheese and Intestinal
Conditions on Macronutrients Digestibility. LWT 2019, 113, 108278. [CrossRef]
6. Nikoo, M.; Regenstein, J.M.; Ahmadi Gavlighi, H. Antioxidant and Antimicrobial Activities of (-)-Epigallocatechin-3-Gallate
(EGCG) and Its Potential to Preserve the Quality and Safety of Foods. Compr. Rev. Food Sci. Food Saf. 2018, 17, 732–753. [CrossRef]
7. Silva, C.C.G.; Domingos-Lopes, M.F.P.; Magalhães, V.A.F.; Freitas, D.A.S.R.; Coelho, M.C.; Rosa, H.J.D.; Dapkevicius, M.L.N.E.
Latin-Style Fresh Cheese Enhances Lactic Acid Bacteria Survival but Not Listeria Monocytogenes Resistance under in Vitro
Simulated Gastrointestinal Conditions. J. Dairy Sci. 2015, 98, 4377–4383. [CrossRef]
8. Andrade, M.A.; Ribeiro-Santos, R.; Guerra, M.; Sanches-Silva, A. Evaluation of the Oxidative Status of Salami Packaged with an
Active Whey Protein Film. Foods 2019, 8, 387. [CrossRef]
9. Cagri, A.; Ustunol, Z.; Ryser, E.T. Antimicrobial Edible Films and Coatings. J. Food Prot. 2004, 67, 833–848. [CrossRef]
10. Ramos, Ó.L.; Fernandes, J.C.; Silva, S.I.; Pintado, M.E.; Malcata, F.X. Edible Films and Coatings from Whey Proteins: A Review on
Formulation, and on Mechanical and Bioactive Properties. Crit. Rev. Food Sci. Nutr. 2012, 52, 533–552. [CrossRef]
11. Díaz-Montes, E.; Castro-Muñoz, R. Edible Films and Coatings as Food-Quality Preservers: An Overview. Foods 2021, 10, 249.
[CrossRef] [PubMed]
12. Siso, M.I.G. The Biotechnological Utilization of Cheese Whey: A Review. Bioresour. Technol. 1996, 57, 1–11. [CrossRef]
13. Mileriene, J.; Serniene, L.; Henriques, M.; Gomes, D.; Pereira, C.; Kondrotiene, K.; Kasetiene, N.; Lauciene, L.; Sekmokiene, D.;
Malakauskas, M. Effect of Liquid Whey Protein Concentrate–Based Edible Coating Enriched with Cinnamon Carbon Dioxide
Extract on the Quality and Shelf Life of Eastern European Curd Cheese. J. Dairy Sci. 2021, 104, 1504–1517. [CrossRef] [PubMed]
14. Pluta-Kubica, A.; Jamróz, E.; Kawecka, A.; Juszczak, L.; Krzyściak, P. Active Edible Furcellaran/Whey Protein Films with Yerba
Mate and White Tea Extracts: Preparation, Characterization and Its Application to Fresh Soft Rennet-Curd Cheese. Int. J. Biol.
Macromol. 2020, 155, 1307–1316. [CrossRef] [PubMed]
15. di Pierro, P.; Sorrentino, A.; Mariniello, L.; Giosafatto, C.V.L.; Porta, R. Chitosan/Whey Protein Film as Active Coating to Extend
Ricotta Cheese Shelf-Life. LWT-Food Sci. Technol. 2011, 44, 2324–2327. [CrossRef]
16. Pluta-Kubica, A.; Jamróz, E.; Juszczak, L.; Krzyściak, P.; Zimowska, M. Characterization of Furcellaran-Whey Protein Isolate
Films with Green Tea or Pu-Erh Extracts and Their Application as Packaging of an Acid-Curd Cheese. Food Bioprocess Technol.
2021, 14, 78–92. [CrossRef]
17. Castro, F.V.R.; Andrade, M.A.; Silva, A.S.; Vaz, M.F.; Vilarinho, F. The Contribution of a Whey Protein Film Incorporated with
Green Tea Extract to Minimize the Lipid Oxidation of Salmon (Salmo salar L.). Foods 2019, 8, 327. [CrossRef]
18. Oliveira, S.P.L.F.; Bertan, L.C.; de Rensis, C.M.V.B.; Bilck, A.P.; Vianna, P.C.B. Whey Protein-Based Films Incorporated with
Oregano Essential Oil. Polimeros 2017, 27, 158–164. [CrossRef]
19. Mosavinezhad, K.; Shakerian, A.; Chaleshtori, R.S.; Rahimi, E. Antimicrobial and Antioxidant Effects of Thymus Daenensis and
Camellia Sinensis Ethanolic Extracts of Chicken Meat During Frozen Storage. J. Med. Plants By-Prod. 2020, 9, 17–27.
20. Prasanth, M.I.; Sivamaruthi, B.S.; Chaiyasut, C.; Tencomnao, T. A Review of the Role of Green Tea (Camellia sinensis) in
Antiphotoaging, Stress Resistance, Neuroprotection, and Autophagy. Nutrients 2019, 11, 474. [CrossRef]
21. Xing, L.; Zhang, H.; Qi, R.; Tsao, R.; Mine, Y. Recent Advances in the Understanding of the Health Benefits and Molecular
Mechanisms Associated with Green Tea Polyphenols. J. Agric. Food Chem. 2019, 67, 1029–1043. [CrossRef] [PubMed]
22. Fakae, L.B.; Stevenson, C.W.; Zhu, X.Q.; Elsheikha, H.M. In Vitro Activity of Camellia sinensis (Green Tea) against Trophozoites
and Cysts of Acanthamoeba Castellanii. Int. J. Parasitol. Drugs Drug Resist. 2020, 13, 59–72. [CrossRef] [PubMed]
23. de Araújo, F.F.; de Paulo Farias, D.; Neri-Numa, I.A.; Pastore, G.M. Polyphenols and Their Applications: An Approach in Food
Chemistry and Innovation Potential. Food Chem. 2021, 338, 127535. [CrossRef] [PubMed]
24. da Silva Pinto, M. Tea: A New Perspective on Health Benefits. Food Res. Int. 2013, 53, 558–567. [CrossRef]
25. Colombo, F.; di Lorenzo, C.; Biella, S.; Vecchio, S.; Frigerio, G.; Restani, P. Adverse Effects to Food Supplements Containing
Botanical Ingredients. J. Funct. Foods 2020, 72, 103990. [CrossRef]
Foods 2022, 11, 1158 22 of 23
26. Kim, H.S.; Quon, M.J.; Kim, J.A. New Insights into the Mechanisms of Polyphenols beyond Antioxidant Properties; Lessons from
the Green Tea Polyphenol, Epigallocatechin 3-Gallate. Redox Biol. 2014, 2, 187–195. [CrossRef]
27. Rubab, S.; Rizwani, G.H.; Bahadur, S.; Shah, M.; Alsamadany, H.; Alzahrani, Y.; Shuaib, M.; Hershan, A.; Hobani, Y.H.; Shah, A.A.
Determination of the GC–MS Analysis of Seed Oil and Assessment of Pharmacokinetics of Leaf Extract of Camellia sinensis L. J.
King Saud Univ.-Sci. 2020, 32, 3138–3144. [CrossRef]
28. Santacruz, S.; Castro, M. Viability of Free and Encapsulated Lactobacillus Acidophilus Incorporated to Cassava Starch Edible
Films and Its Application to Manaba Fresh White Cheese. LWT 2018, 93, 570–572. [CrossRef]
29. Jutinico-Shubach, A.; Gutiérrez-Cortés, C.; Suarez, H. Antilisterial Activity of Chitosan-Based Edible Coating Incorporating
Cell-Free Supernatant from Pediococcus Pentosaceus 147 on the Preservation of Fresh Cheese. J. Food Process. Preserv. 2020,
44, e14715. [CrossRef]
30. Ribeiro-Santos, R.; de Melo, N.R.; Andrade, M.; Azevedo, G.; Machado, A.V.; Carvalho-Costa, D.; Sanches-Silva, A. Whey Protein
Active Films Incorporated with a Blend of Essential Oils: Characterization and Effectiveness. Packag. Technol. Sci. 2018, 31, 27–40.
[CrossRef]
31. Nickavar, B.; Esbati, N. Evaluation of the Antioxidant Capacity and Phenolic Content of Three Thymus Species. JAMS J. Acupunct.
Meridian Stud. 2012, 5, 119–125. [CrossRef] [PubMed]
32. Moon, J.K.; Shibamoto, T. Antioxidant Assays for Plant and Food Components. J. Agric. Food Chem. 2009, 57, 1655–1666.
[CrossRef] [PubMed]
33. Amorati, R.; Valgimigli, L. Advantages and Limitations of Common Testing Methods for Antioxidants. Free Radic. Res. 2015, 49,
633–649. [CrossRef] [PubMed]
34. Prieto, M.A.; Rodríguez-Amado, I.; Vázquez, J.A.; Murado, M.A. β-Carotene Assay Revisited. Application to Characterize and
Quantify Antioxidant and Prooxidant Activities in a Microplate. J. Agric. Food Chem. 2012, 60, 8983–8993. [CrossRef] [PubMed]
35. Ueno, H.; Yamakura, S.; Arastoo, R.S.; Oshima, T.; Kokubo, K. Systematic Evaluation and Mechanistic Investigation of Antioxidant
Activity of Fullerenols Using β -Carotene Bleaching Assay. J. Nanomater. 2014, 2014. [CrossRef]
36. Andrade, M.A.; Ribeiro-Santos, R.; Costa Bonito, M.C.; Saraiva, M.; Sanches-Silva, A. Characterization of Rosemary and Thyme
Extracts for Incorporation into a Whey Protein Based Film. LWT 2018, 92, 497–508. [CrossRef]
37. Bondet, V.; Brand-Williams, W.; Berset, C. Kinetics and Mechanisms of Antioxidant Activity Using the DPPH • Free Radical
Method. LWT-Food Sci. Technol. 1997, 30, 609–615. [CrossRef]
38. Kedare, S.B.; Singh, R.P. Genesis and Development of DPPH Method of Antioxidant Assay. J. Food Sci. Technol. 2011, 48, 412–422.
[CrossRef]
39. Škrovánková, S.; Mišurcová, L.; Machů, L. Antioxidant Activity and Protecting Health Effects of Common Medicinal Plants. Adv.
Food Nutr. Res. 2012, 67, 75–139.
40. Blois, M.S. Antioxidant Determinations by the Use of a Stable Free Radical. Nature 1958, 181, 1199–1200. [CrossRef]
41. P˛ekal, A.; Pyrzynska, K. Evaluation of Aluminium Complexation Reaction for Flavonoid Content Assay. Food Anal. Methods 2014,
7, 1776–1782. [CrossRef]
42. Ahmed, F.; Iqbal, M. Antioxidant Activity of Ricinus Communis. Org. Med. Chem. Int. J. 2018, 5, 107–112. [CrossRef]
43. Pontis, J.A.; Costa, L.A.M.A.D.; Silva, S.J.R.D.; Flach, A. Color, Phenolic and Flavonoid Content, and Antioxidant Activity of
Honey from Roraima, Brazil. Food Sci. Technol. 2014, 34, 69–73. [CrossRef]
44. Silva, L.; Pezzini, B.; Soares, L. Spectrophotometric Determination of the Total Flavonoid Content in Ocimum basilicum L.
(Lamiaceae) Leaves. Pharmacogn. Mag. 2015, 11, 96. [CrossRef] [PubMed]
45. Yoo, K.M.; Lee, C.H.; Lee, H.; Moon, B.K.; Lee, C.Y. Relative Antioxidant and Cytoprotective Activities of Common Herbs. Food
Chem. 2008, 106, 929–936. [CrossRef]
46. Gulcin, İ. Antioxidants and Antioxidant Methods: An Updated Overview. Arch. Toxicol. 2020, 94, 651–715. [CrossRef]
47. Singleton, V.L.; Rossi, J.A. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am. J. Enol.
Vitic. 1965, 16, 144.
48. Kodama, D.H.; Gonçalves, A.E.D.S.S.; Lajolo, F.M.; Genovese, M.I. Flavonoids, Total Phenolics and Antioxidant Capacity:
Comparison between Commercial Green Tea Preparations. Food Sci. Technol. 2010, 30, 1077–1082. [CrossRef]
49. Almeida, T.S.D.; Araújo, M.E.M.; Rodríguez, L.G.; Júlio, A.; Mendes, B.G.; Santos, R.M.B.D.; Simões, J.A.M. Influence of
Preparation Procedures on the Phenolic Content, Antioxidant and Antidiabetic Activities of Green and Black Teas. Braz. J. Pharm.
Sci. 2019, 55. [CrossRef]
50. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. [14] Analysis of Total Phenols and Other Oxidation Substrates and
Antioxidants by Means of Folin-Ciocalteu Reagent. In Methods in Enzymology; Academic Press: Cambridge, MA, USA, 1999;
Volume 299, pp. 152–178.
51. Komes, D.; Horžić, D.; Belščak, A.; Ganić, K.K.; Vulić, I. Green Tea Preparation and Its Influence on the Content of Bioactive
Compounds. Food Res. Int. 2010, 43, 167–176. [CrossRef]
52. Lachman, J.; Hosnedl, V.; Pivec, V.; Orsák, M. Polyphenols in Cereals and Their Positive and Negative Role in Human and Animal
Nutrition. In Proceedings of the Cereals for Human Health and Preventive Nutrition, Brno, Czech Republic, 7–11 July 1998;
pp. 118–125.
53. Jakubczyk, K.; Kochman, J.; Kwiatkowska, A.; Kałduńska, J.; Dec, K.; Kawczuga, D.; Janda, K. Antioxidant Properties and
Nutritional Composition of Matcha Green Tea. Foods 2020, 9, 483. [CrossRef] [PubMed]
Foods 2022, 11, 1158 23 of 23
54. Martins, C.; Vilarinho, F.; Sanches Silva, A.; Andrade, M.; Machado, A.V.; Castilho, M.C.; Sá, A.; Cunha, A.; Vaz, M.F.; Ramos, F.
Active Polylactic Acid Film Incorporated with Green Tea Extract: Development, Characterization and Effectiveness. Ind. Crops
Prod. 2018, 123, 100–110. [CrossRef]
55. Chalob, K.K.; Abdul-Rahman, S.M. Antimicrobial Activity of Edible Film from Whey Protien Isolate Incorported with Green Tea
Extract and Its Use in Cheese Coatin. Iraq J. Mark. Res. Consum. Prot. 2018, 10, 50–60.
56. García-Díez, J.; Saraiva, C. Use of Starter Cultures in Foods from Animal Origin to Improve Their Safety. Int. J. Environ. Res.
Public Health 2021, 18, 2544. [CrossRef]
57. Lourenço, S.C.; Moldão-Martins, M.; Alves, V.D. Antioxidants of Natural Plant Origins: From Sources to Food Industry
Applications. Molecules 2019, 24, 4132. [CrossRef]

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1 Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] (J.R.);

Publisher’s Note: MDPI stays neutral

Foods 2022, 11, 1158. https://doi.org/10.3390/foods11081158 https://www.mdpi.com/journal/foods

2. Materials and Methods

2.2. Preparation of Green Tea Infusions

2.3. Extraction of Green Tea Extracts

2.3.2. Solid–Liquid Extraction with Soxhlet Apparatus

2.4. Preparation of Whey-Based Protein Films

2.5. Evaluation of the Antioxidant Properties

2.5.1. β-Carotene Bleaching Assay

2.5.2. DPPH Radical-Scavenging Method

Absorbance was measured at 515 nm using a Hitachi U-3900 spectrophotometer(Tokyo,

2.5.3. Determination of Total Flavonoid Content (TFC)

2.5.4. Determination of Total Phenolic Compounds (TPC)

2.6. Fresh Cheese Samples

2.7. Evaluation of the Lipid

2.7. Evaluation of the Lipid Oxidation Status (TBARS Assay)

2.8. Microbiological Analysis

packages, various parameters were determined: total microorganisms at 30 ◦ C according

2.9. Statistical Analysis

3. Results and Discussion

Foods 2022, 11, 1158 0 9 of 23

β-carotene bleaching inhibition test

Total phenolic compounds

DPPH radical inhibition test

TEAC (μg TE/ml tea)

3.1.2. Evaluation of the Antioxidant Capacity of Green Tea Extracts

Happy Flora (Soxhlet)

Happy Flora (Conventional)

0 500 1000 1500 2000

Happy Flora (Soxhlet)

Happy Flora (Conventional)

0 200 400 600 800 1000

3.2. Antioxidant and Antimicrobial Properties of the Active Film

3.2.1. Evaluation of the Antioxidant Capacity of the Whey Protein-Based Films

3.2.1. Evaluation of the Antioxidant Capacity of the Whey Protein-Based Films

Camellia sinensis extract

Foods 2022, 11, x FOR PEER REVIEW 17 of 26

Camellia sinensis extract

0 50 100 150 200

Camellia sinensis extract

0 50 100 150 200

Camellia sinensis extract

3.2.3. Microbiological Analysis of Packaged Fresh Cheeses

Foods 2022, 11, x FOR PEER REVIEW 20 of 26

Goat Fresh Cheese

Goat Fresh Cheese (Blank)

Goat Fresh Cheese (Active)

Sheep and Goat Fresh Cheese

0 5×10 6 1×10 7 1.5×10 7

Goat Fresh Cheese (Active)

Goat Fresh Cheese (Blank)

Goat Fresh Cheese

0 5×10 1 1×10 2 1.5×10 2 2×10 2 2.5×10 2

Sheep and Goat Fresh Cheese (Active)

Sheep and Goat Fresh Cheese (Active)

Sheep and Goat Fresh Cheese (Blank)

Sheep and Goat Fresh Cheese