National Journal of Advanced Research

www.multidisciplinaryarticle.in
ISSN: 2455-216X
Received: 03-04-2024, Accepted: 24-04-2024, Published: 18-05-2024
Volume 10, Issue 1, 2024, Page No. 69-77

Improving the quality of fresh artisan cheeses by encapsulating thyme essential oil with LPS
activation
Dely M*, Ben Haj Koubair H, Mnasser H, Mankai M
Department of Food Technology, University of Carthage, Higher Institute of Food Industries of Tunisia, ESIAT, Tunisia

Abstract
The aim of this study was to compare the effect of combining the encapsulation of essential oil with lactoperoxidase activation
as a decontamination tool. Microbiological, physico-chemical (titratable acidity, pH, total dry extract, fat, fat-free dry extract,
moisture and firmness) and organoleptic analyses were carried out during storage of the cheeses at +4±1ºC for 18 days, and the
the shelf life was determined.
The use of encapsulation of essential oil with lactoperoxidase activation had a significant effect on microbiological quality
compared with control cheeses and those activated by lactoperoxidase alone throughout the shelf life.
The combination of essential oil of thyme encapsulated with lactoperoxidase showed that on the first day, the different
concentrations had a significant effect on the microbiological quality compared with the control and lactoperoxidase cheeses,
but after nine days it was noted that only the HEt concentrations of 1.5 and 1ml/kg had a significant impact on the
microbiological quality of the cheese compared with the other samples.
However, the activation of lactoperoxidase alone had no significant impact on the development of these germs.
With regard to sensory analysis, no descriptor had a significant effect detected by the panel throughout the shelf life.
On the other hand, these conservation combination enabled the shelf life to be extended. The control cheese had a use-by date
of 3.63 days and the cheese preserved with lactoperoxidase only 3.09 days. On the other hand, the combination of thyme
essential oil encapsulated with LPS increased the best-before date of the fresh cheese by 4.19, 8.01 and 10.40 days respectively
for HEt concentrations of 0.5, 1 and 1.5 ml/kg with LPS activation.

Keywords: Microbiological quality, essential oil encapsulations, thyme, organoleptic quality

Introduction This makes the application of barrier technology very

Cheese is generally a nutrient-dense and well-tolerated interesting as it can extend shelf life while preserving
fermented dairy product consumed worldwide. However, nutritional and sensory value. This technology works at
the health effects of cheese consumption remain a matter of lower temperatures and with shorter processing times, while
controversy (Zhang et al., 2023) [30] ensuring microbiological safety (Predrag Putnik et al., 2020)
[24]
On one hand, cheese is a rich source of high-quality protein . The combination technique involves preserving food
(mainly casein), lipids, minerals (e.g., calcium, phosphorus, using several agents/methods applied simultaneously or
and magnesium), and vitamins (e.g., vitamin A, K2, B2, sequentially (Peleg, 2020) [22].
B12, and folate), and probiotics and bioactive molecules The combination of different technologies and/or the
(e.g., bioactive peptides, lactoferrin, short-chain fatty acids, adjustment of a food's pH, water activity (aW), oxygen
and milk fat globule membrane), which may provide various tension, etc., can be used to improve its preservation
health benefits (Zhang et al., 2023) [30] efficiency, which has been widely documented and has
On the other hand, cheese has a fairly short shelf life due to become common knowledge (Peleg, 2020) [22].
its multiple varieties of micro-organisms such as yeasts, The latter is widely used in industrialised as well as
Pseudomonas sp, heterofermentative lactic bacteria, developing countries for efficient food preservation (Peng et
Clostridia, Bacillus sp, coliforms, Klebsiella pneumoniae, al.,2015) [23].
Penicillium and psychrophilic spoilage microflora (Zhu et Combination technology is an important approach that can
al., 2020) [31]. be used to improve quality parameters during food
The application of heat treatments at very high temperatures processing and storage. Intelligent application of barriers
can alter the composition of milk as it affects protein improves sensory characteristics, food chemical and
structures and water-soluble vitamins, resulting in a microbiological qualities (Peng et al., 2015) [23]. Moreover,
decrease in total fat and total solids and an increase in urea more than 60 reported barriers are available, these can be
(Bezie, 2019) [5]. used in different combinations and concentrations for a wide
Alternative processes are therefore needed to solve this range of foods. This versatility makes the application of the
problem, mainly the use of combination technology (Peng et technology possible in modern and local food processing
al., 2015) [23]. (Peng et al., 2015) [23].
At the crossroads of major economic, environmental and A particular example is the use of lactoperoxidase, a natural
public health issues, food preservation techniques today enzyme considered to be an important element in the host's
deserve the mobilisation of all stakeholders to ensure that natural defence system against bacterial infection. Indeed,
technologies evolve towards solutions that are more the LPO system can also be used to increase the stability of
respectful of both the environment and the consumer milk storage at high ambient temperatures (Zarei et al.,
(Gontard et al., 2017) [10]. 2016) [28].

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Non-thermal processes are implemented to inactivate 0.125 g/l of mesophilic starter culture (Barukcic et al.,
spoilage micro-organisms and improve the nutritional, 2020).
sensory and microbial characteristics of the product; a good Fermentation (acid coagulation) of the milk was carried out
opportunity that presents itself is the use of essential oils at 30 C for about 16 h until curd was formed and pH of
during cheese processing (Aprotosoaie et al., 2010) [3]. about 4.6 was reached (Barukcic et al., 2020).
The food industry uses essential oils to enhance the flavour At this point, the fermentation process was interrupted by
and colour of foods. In addition, essential oils have different rapid cooling. The resulting curd was cut by a sterile knife
chemical composition profiles, enabling them to be used as and heated to a maximum of 40°C while continuing
natural food preservatives (Aprotosoaie et al., 2010) [3]. agitation to allow separation of the whey. Transferred to
Encapsulation is one of the techniques commonly used. It molds in cold storage (15-16°C) and drained (Barukcic et
enables the volatile compounds in essential oils to be al., 2020).
immobilised, stabilising the oil and protecting it from light, The cheese yield is calculated according to the following
oxygen and temperature, as well as modulating its release formula:
by prolonging its kinetic profile (Kerdudo et al., 2015) [12].
Consequently, this process tends to protect and preserve the
biological activities of these oils. In addition, putting these
virtues into action in the food product (Kerdudo et al., 2015)
[12] Y: means the cheese yield in %.
.
The aim of encapsulation is to ensure the protection, Wc: means the weight of cheese obtained in kilograms.
compatibility and stabilisation of an active ingredient in a W: means the weight of the milk in kilograms.
formulation. It can improve the presentation of a product or
mask an odour or taste (Akdim, 2016) [2]. LPS activation
The sample was subjected to refrigeration combined with
It can also modify and control the release profile of an
active substance to obtain, for example, a prolonged or activation of the LP system by addition of sodium
triggered effect (Akdim, 2016) [2]. thiocyanate (NaSCN) as a source of thiocyanate (SCN) to a
final concentration of 14 mg⁄L (Boulares et al., 2011) [7].
Materials and methods After 1 min of thorough mixing of the milk, 30 mg ⁄L of
Harvesting and drying sodium percarbonate (2Na2CO33H2O) was added as a
source of hydrogen peroxide (H2O2) as recommended by the
The freshly harvested plant material (thyme) is cleaned of
weeds, dried in the shade in an airy, dry place away from International Dairy Federation (IDF1988).
light and pollution, then cut into very fine pieces.
Encapsulation of essential oils
Extraction Encapsulation is a potentially beneficial procedure for the
Steam stripping protection and proper preservation of essential oils against
degradation processes (oxidation and hydrolysis), as well as
This is one of the official methods for obtaining EO. In this
extraction system, the plant material is subjected to the for stabilizing the release of high-value compounds
action of a stream of steam without prior maceration. The extracted from fruits, vegetables, and waste products (Le
Priol, 2021) [14].
vapours, saturated with volatile compounds, are condensed
and then decanted into the essencier, before being separated The sodium alginate gel is poured very gradually to form
into an aqueous phase (HA) and an organic phase (EO) the essential oil bead, and each bead remains in the calcium
(Nadjib et al., 2019) [21]. chloride solution for about 30min (Le Priol, 2021) [14]. After
The absence of direct contact between the water and the the recovery of the EO capsules, dry them by absorbent
paper, store the capsules in cold (4°C) in sterile petri dishes
plant material, and then between the water and the aromatic
molecules, avoids certain hydrolysis or degradation (Le Priol, 2021) [14].
phenomena that could harm the quality of the oil. In
The samples were distributed as follows
addition, the fragrance of the EO obtained is more delicate
and distillation, which is regular and faster, means that the
LPS+ 0,5 LPS+ 1 LPS+ 1,5
top notes are rich in esters (Nadjib et al., 2019) [21]. Samples Control LPS
ml/kg ml/kg ml/kg
Moreover, according to the AFNOR standard (1986), the All the tests are spread over three periods: t0, t9 and t18.
essential oil yield is defined as the ratio between the mass of
the essential oil obtained and the mass of the plant material Physicochemical analysis
used. The yield expressed as a percentage is calculated by pH
the following formula The pH is measured with a pH meter (KDD002) to 0.01
units of accuracy.
For cheese, the electrode of the pH meter is placed directly
in the cheese (Fedala et al., 2020) [9]
Y: yield of essential oil ;
W1: weight of essential oil obtained in g; Titratable acidity
W2: weight of thyme in g This is an acid-base titration, lactic acid is neutralized by a
solution of sodium hydroxide NaOH in the presence of
Cheese manufacture phenolphthalein as a colored indicator ((Fedala et al., 2020)
The cheese was produced from raw cow’s milk. [9]
.
Subsequently, the standardized milk was pasteurized at For milk the acidity is expressed in degree Dornic (°D).
85°C/10 min, cooled to 32 C, and inoculated by adding The titration is performed by an alkaline solution (NaOH,

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N/9) in the presence of phenolphthalein at 1% (w/v), (1 mL Sensory analysis

of NaOH (N/9) corresponds to 0.01 g of lactic acid per cent) Once the production is finished, a hedonic test is carried out
(Fedala et al., 2020) [9]. for the two cheeses. The purpose of this test is to compare
the overall hedonic appreciation of the different cheeses by
Determination of fat focusing on the individual feelings related to the pleasure or
The method is based on the acid-butyrometric method of displeasure caused by the food (Fedala et al., 2020) [9].
Van Gulik. It is based on the dissociation of cheese proteins Consumer acceptance was determined using a 9-point scale.
by the addition of sulfuric acid and separation of the fat by The results of the sensory analysis are represented by a
centrifugation in a Van Gulik butyrometer. radar graph. It allows to highlight the data around a
In a Van – Gulik butyrometer, put 3g of cheese, add sulfuric reference value (Fedala et al., 2020) [9].
acid so that it covers the cheese mass, making the proteins
dissociate in a water bath. After complete dissociation, fill Microbiological analysis
the graduated rod with sulfuric acid and add 1 ml of isoamyl The cheese was aseptically removed from the package and
alcohol (Fedala et al., 2020) [9]. The separation of fat is done the surface was cut with a sterile knife. The cheese slices
by centrifugation (Fedala et al., 2020) [9]. were weighed into a sterile beaker with 100 ml of sterile
The fat expressed in g/100g of cheese is obtained by direct distilled water. The cheese was grated and homogenized
reading on the butyrometer scale (Fedala et al., 2020) [9] thoroughly (Fedala et al., 2020) [9]
Serial dilutions of the homogenates were placed on
Determination of total dry extract appropriate media in Petri dishes and analyzed immediately
The total dry extract (TDE) is determined using an oven set (Fedala et al., 2020) [9]. The numbers of total coliforms, total
at 103±2°C. The test samples are measured to the nearest 1 aerobic mesophilic flora, yeasts and molds were monitored
mg and the dry matter is expressed as a percentage by on consecutive days during storage (Fedala et al,. 2020) [9]
weight by the remainder after drying (Fedala et al., 2020) [9]. The media and incubation conditions used were as follows:
Two grams of cheese are weighed after homogenization of Coliforms: spread on violet red bile agar (VRBA), with a
the cheese paste, and the dry matter is expressed as weight double cover layer of the same medium, incubated at 30° C
percentage by the remainder after drying (Fedala et al., for 24 to 48 hours (Fedala et al,. 2020) [9]
2020) [9]. Total aerobic mesophilic flora: spread on PCA (Plate Count
Agar) and incubation is done at 30°C for 48 hours (Fedala et
al,. 2020) [9]
Yeasts and molds: spread on Sabouraud dextrose agar,
TDE: total dry extract incubated at 25° C for 5 to 7 days (Fedala et al,. 2020) [9]
E1: weight in grams of the capsule and the test sample.
E2: weight in grams of the capsule and residue after drying The determination of shelf life
and cooling. The general equation that describes the loss of quality of a
Mc: weight in grams of the empty capsule. food is applicable for any factor A is as follows:

Determination of the dry defatted extract

It is the result of the difference between the total dry extract
and the fat content and r: rate of the degradation reaction = rate of formation of A ;
determined as follows: K: reaction rate constant or apparent rate ;
A: concentration of the factor to be followed;
n: order of the degradation reaction.

TDE: total dry extract Statistical analysis

ESD: dry extract defatted All experiments were repeated at least three times. The
MG: Fatty matter results were subjected to a one-factor analysis of variance
with a significance level of 95% using Excel for the radar
Measurement of moisture presented for the sensory analysis and by SPSS software
The moisture is calculated by the following formula: using the one-factor ANOVA test.

Results and disscusion

Thyme essential oil extraction yield
Rheological study According to the formula defined by the AFNOR standard
Hardness (N), of cheese was determined by texturometer (1986), we obtained a yield in OEt: Y= 1.96%.
analyzer type TVT 6700. Cheese samples were cut into 5 * Sahraoui et al, (2016) [25] have shown that the yield of
5 cm cubes and stored at a room temperature before essential oil obtained by steam stripping varies between
1.50% and 2.96%.
measurements (Barukcic et al., 2020). This variability in essential oil, both in terms of composition
Then the prepared samples were subjected to double and yield between these plants, can be explained by various
compression at a traverse speed of 1 mm/s and a penetration factors of intrinsic origin, specific to the plant's genetic or
distance of 40 mm up and down with 10 s between the two extrinsic, linked to the plant's growth and development
cycles (Barukcic et al.,2020) conditions (Sahraoui et al., 2016) [25].

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We would also stress the importance of choosing the thyme According to our results, we obtained a Cheese yield Y=
harvesting period in order to obtain the quality and quantity 10.7%.
of oil. Yields generally differ from one period to another
(Sahraoui et al., 2016) [25]. Physicochemical analyses
Effect of Hurdle technology on pH
Cheese yield No significant difference (P > 0.05) in pH was observed
Cheese yield is of great interest in the cheese industry between the control and the LPS-activated cheese. Seifu et
because it reflects the overall quantitative distribution of al, (2004) [26] stated that activation of cheese by LPS had no
milk constituents during draining (Vignola et al., 2002) [27]. effect on pH. Similarly, no significant difference in pH was
It requires a high level of dry extract and more specifically observed between the controls and the cheeses activated by
protein (casein) and high fat concentrations (Vignola et al., LPS throughout the ripening period (Boulares et al., 2011)
2002) [27]. [7]
.

Table 1: Effect of thyme essential oil encapsulation with LPS activation on fresh cheese physicochemical parameters
Fat (g/kg)
pH Acidity (°D) Total dry extract (g/kg) Defatted dry extract (g/kg) Moisture (%)
t0 : t0 : t0 : t0 : t0 :
t0 : 35,73±0,21
4,58±0,01 14,53±0,06 30,50±0,26 5,23±0,23 64,27±0,21
t9 : t9 : t9 : t9 : t9 : t9 :
Control
4,34±0,04 16,43±0,06 29,60±0,17 34,83±0,06 5,23±0,15 65,17±0,06
t18 : t18 : t18 : t18 : t18 : t18 :
4,25±0,02 17,17±0,12 28,70±0,36 34,20±0,17 5,60±0,53 65,90±0,00
t0 : t0 : t0 : t0 : t0 :
t0 : 35,17±0,25
4,56±0,01 14,77±0,06 30,80±0,1 4,33±0,25 64,83±0,25
t9 : t9 : t9 : t9 : t9 : t9 :
LPS
4,32±0,01 16,73±0,06 30,23±0,42 34,43±0,06 4,27±0,25 65,50±0,17
t18 : t18 : t18 : t18 : t18 : t18 :
4,22±0,01 17,57±0,12 29,17±0,12 33,90±0,1 4,73±0,06 66,10±0,1
t0 : t0 : t0 t0 :
t0 : 4,53±0,01 t0 : 36,10±0,2
15,53±0,12 31,27±0,21 4,83±0,15 63,90±0,2
LPS t9 : t9 : t9 : t9 : t9 : t9 :
+ 4,51±0,01 15,33±0,06 30,70±0,26 35,90±0,1 5,20±0,36 64,10±0,1
0,5ml/kg t18 : t18 : t18 : t18 : t18 : t18 :
4,48±0,01 15,67±0,12 30,20±0,17 35,37±0,38 5,17±0,47 64,63±0,38
t0 : t0 : t0 : t0 :
t0 : 4,51±0,01 t0 : 36,70±0,17
15,77±0,06 31,57±0,15 5,13±0,12 63,30±0,17
LPS t9 : t9 : t9 : t9 : t9 : t9 :
+ 4,50±0,01 15,73±0,06 30,77±0,49 35,93±0,06 5,17±0,47 64,07±0,06
1 ml/kg t18 : t18 : t18 : t18 : t18 : t18 :
4,47±0,01 15,97±0,12 29,87±0,21 35,13±0,06 5,27±0,23 64,87±0,06
t0 : t0 : t0 : t0 : t0 :
t0 : 37,10±0,1
4,48±0,01 15,97±0,12 31,90±0,2 5,53±0,38 62,57±0,21
LPS t9 : t9 : t9 : t9 : t9 : t9 :
+ 4,46±0,01 15,93±0,15 31,60±0,1 36,77±0,15 5,17±0,06 63,23±0,15
1,5ml/kg t18 : t18 : t18 : t18 : t18 : t18 :
4,45±0,01 16,33±0,12 31,03±0,38 36,30±0,17 5,27±0,47 63,70±0,17

Table 1 shows the effect of the pH of the cheese at t0, t9 and particular the lactic acid bacteria responsible for the
t18 of storage at 4±1°C after a combination of treatment with acidification of the cheese.
the incorporation of the encapsulated essential oil at On the other hand, at t9 and t18, the different combinations of
different concentrations with 0.5, 1 and 1.5 ml /kg and the essential oil and LPS had no significant effect on the pH of
activation of LPS compared with a control and a sample the cheese, which resulted in pH stability.
activated only with LPS. Messouadi et al, (2019) found that the combination of LPS
In fact, the average pH of the control was 4.58 ± 0.01 at t0 and essential oil preserved the pH of raw cow's milk during
and decreased over time to reach a value of 4.25 ± 0.01 at storage.
the end of storage. This allows the use of this combination technique to be
The pH values were found to be proportional to the high prioritised since the decrease in pH in foods could adversely
concentrations of essential oil added to the cheeses, affect sensory quality, resulting from metabolic degradation
decreases from 4.54 ± 0.01 to 4.51 ± 0.01 and 4.48 ± 0.01 by microorganisms, particularly in carbohydrate-rich foods,
for the doses of 0.5, 1 and 1.5ml/kg added respectively to which can be used by microorganisms to produce acids
(Cheon et al., 2016).
the products at t0.
Following the addition of the essential oils combined with Effect of Hurdle technology on acidity
LPS, we recorded a significant decrease in pH compared The acidity developed in cheese results from the
with the control and those activated by LPS can be transformation of lactose into lactic acid. It is measured by
attributed to the inhibition of the microbial flora and in titration (Gadi et al., 2020). The low titratable acidity values

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recorded in our results reflect weak lactic fermentation in Similarly, for the cheese that had only undergone LPS n
the cheese samples, which depends on the casein, mineral activation, the average dry extract decreased from 35.17
salt and ion content. It also depends on the hygienic g/kg at t0 to 33.9 g/kg at t18.
conditions during milking, the total microbial flora and its On the other hand, the total dry extract increased slightly for
metabolic activity and the handling of the milk (Gadi et al., the cheeses treated with the hurdle technique compared with
2020). the controls and those activated by LPS.
Table 1 shows the effect of cheese acidity at t0, t9 and t18 of In fact, the decrease in total dry extract over time is due to
storage at 4±1°C after a combination of treatment with the the liquefaction of the product Messaour (2018) [19].
incorporation of encapsulated essential oil at different
concentrations of 0.5, 1 and 1.5 ml/kg and LPS activation Effect of Hurdle technology on fat content
compared with a control and a sample activated only with Fat content and dry matter are very important in cheese
LPS. production because milk lipids are characterised by the
Also, these values increase significantly with the doses of presence of relatively short-chain fatty acids which can be
Thyme incorporated into the products by encapsulation absorbed by a simpler mechanism than long-chain fatty
compared to that of the control and that activated only by acids. Milk fat plays an essential role in the development of
LPS. both the taste and texture of cheese (Vignola, 2002) [27].
These values increase from 14.53 to 14.77 to 15.53 to 15.77 Cheese texture depends on its fat content. In fact, the water
and to 15.97°D on average for Thyme concentrations content and the proportions of long polysaturated fatty acids
varying from 0, LPS, 0.5; 1; 1.5ml/kg respectively, in the in the milk determine the texture of the dough: extra hard,
cheeses. semi-soft, soft, and so on. For example, with more than 60%
Lactic acid bacteria ferment lactose and acidify milk fat and less than 51% water, you get an extra-hard cheese.
through the massive production of lactic acid (Leksir, 2018). Too high a fat content can lead to problems with draining
The growth of lactic acid bacteria in the milk, then in the and coagulation (Vignola, 2002) [27].
curd, leads to the consumption of lactose and the excretion Table 1 shows that the control cheese samples had a fat
of lactic acid, lowering the pH. This acidifying function of content of 30.5 g/kg and that activated by LPS 30.8 g/kg,
lactic acid bacteria is decisive in the cheese-making process while the cheese treated with the combination of activated
(Leksir, 2018). LPS essential oil had an average of 31.27 g/kg, 31.57 g/kg
The increase in titratable acidity is mainly due to proteolysis and 32.03 g/kg respectively.
and the decarboxylation of amino acids released by This technique had a significant effect on fat content, with a
microorganisms accumulated during product preservation significant increase in the cheese with the highest
(Leksir, 2018). concentration combined with LPS compared with all the
Usually, pH and acidity are inversely proportional. It is other samples at t0, t9 and t18.
suggested that this proportionality is due to the activity of This was approved by Messouadi et al, (2019) showing that
the lactic flora, which is fairly high, or that the environment the activation of the LPS system combined with the addition
does not allow the development of lactic acid degradation of juniper and Phoenicia essential oils have a significant
flora in large numbers (Leksir, 2018). effect on the variation of the fat content of cow's milk.
On the other hand, any increase in acidity favours the On the other hand, LPS activation had no significant effect
solubilisation of minerals and the destabilisation of casein on the variation in fat content compared with the controls.
micelles, leading to excessive losses in the whey during Similarly, the other two concentrations 0.5 and 1ml/kg
processing, which has a direct influence on the quality of combined with LPS have no significant effect compared
our cheeses (Leksir, 2018). with the controls and those activated by LPS throughout the
shelf life.
Effect of Hurdle technology on total dry extract According to Bellivier et al, (2000) [6], the lipid content of
The level of dry extract varies from one type of cheese to milk intended for cheese production largely determines the
another. This difference is due to the use of salt and the fat content of the finished product.
draining time. The cheese's high dry matter content gives it In fact, the fat content of cheese depends solely on the
a relatively firm consistency. nature and initial composition of the milk used to make it.
Table 1 illustrates the variation in the total dry extract According to Morgan el al (2001) [20], cheese fats are a good
content of cheese treated with different concentrations of source of energy and are generally easy to digest (88% to
essential oil combined with LPS as well as cheeses (control 94%).
and only LPS-activated) during 18 days of refrigerated
storage at 4 ± 1°C and tested on days 0, 9 and 18. Effect of Hurdle technology on defatted dry extract
Throughout the storage period, there was a significant Table 1 shows the variation in fat-free dry extract of treated
increase in the total dry extract of the control and LPS- and untreated cheeses stored at 4±1°C. It can be seen that
activated cheeses compared to the cheeses that had the defatted dry extract values increased proportionately,
undergone the hurdle treatment. rising from 4.83, 5.13 and 5.53 g/kg at t0 and remaining
This result is in agreement with that of Messaour (2018) [19]. non-significant.
The total dry extract is around 43.26% for the 0.1%, 45.94% On the other hand, we note that all the samples that
for the 0.5% and 48.86% for the 1%, this small difference is underwent the hurdle combination had a significant effect
probably due to the addition of the essential oil. compared to the cheeses activated only by LPS at t9, and we
It can be seen that the loss of total dry extract increases over note that the combination with the 1.5ml/kg concentration
time. In fact, the control cheeses had an average total dry with LPS had a significant effect compared to the control
extract of 35.73 g/kg, falling to 34.2 g/kg after 18 days at cheeses.
4±1°C.

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This result is in agreement with that of Messouadi et al, The germs sought and counted in our work are considered to
(2019) showing that the activation of the LPS system be indicators of the overall quality of the finished product
combined with the addition of the essential oils of juniper and reflect compliance or non-compliance with good
oxycèdre and Phénicie has a significant effect on the hygiene practices (Gadi et al., 2020).
variation of the defatted dry extract of cow's milk.
Total coliforms
Effect of Hurdle technology on humidity Figure 2 shows a total absence of total coliforms directly
For the moisture parameter, if the values are outside the after treatment (t0) with all doses combined with LPS. Even
standards, there is a risk of having a fairly hard dough (if the with LPS activation alone, there was a reduction in
moisture content is less than 58%), or a fairly moist, and proliferation compared with the control.
therefore fragile, dough if the moisture content% is greater In fact, at t0, there was a significant decrease in the
than 60%. The TSE content is linked to the moisture evolution of total coliforms with all the concentrions of
content%, so outside the range given in table (40-42), there essential oils combined with LPS compared with the control
is a risk of varying the moisture content% and therefore cheeses and those activated by LPS. On the other hand, LPS
ultimately contributing to the hardness or brittleness of the activation significantly reduced the evolution of total
dough. coliforms.
Table 1 shows that the moisture content of cheese samples On the other hand, at t9, there was a significant decrease in
with encapsulated thyme essetille oil was significantly lower the evolution of total coliforms in the cheese with the
than that of control samples and LPS-activated cheeses at t0. highest concentration of essential oils (1.5ml/kg) combined
Indeed for the concentration of 1.5ml/kg combined with with LPS compared with the control and activated by LPS
LPS, there was a significant difference compared to all other only, and the other two samples.
samples, as well as for the cheese with encapsulated There was also a significant decrease in the evolution of
essential oil concentration 1.5ml/kg with LPS, there was a total coliforms in the cheese with an essential oil
significant effect of moisture compared to control cheeses concentration of 1ml/kg combined with LPS compared to
and those only with LPS throughout the shelf life. The the control. The same results were obtained at t18 as at t9.
difference in moisture content may be due to the water
composition of the milk.

Rheological study
Texture is an essential factor in consumer acceptance of a
product. The term 'firmness' is commonly used to describe a
parameter assessed by means of empirical mechanical tests
and considered as an attribute that must be maintained
during storage and processing.

Fig 2: Effect of encapsulated thyme essential oil incorporation on

total coliforms in fresh cheese (Control; LPS; 0.5ml/kg +LPS; 1
ml/kg +LPS; 1.5ml/kg+LPS)

Mesophilic aerobes
The number of total aerobic mesophilic flora in cheese can
cause premature swelling and the production of enterotoxins
in cheese (Seifu et al., 2004) [26].
Figure 2 shows the effect of the incorporation of
Fig 1: Effect of encapsulated thyme essential oil incorporation on encapsulated thyme essential oil combined with LPS on the
fresh cheese firmness (Control ; LPS ; 0.5ml/kg +LPS ; 1 ml/kg total mesophilic aerobes of fresh cheese compared with
+LPS ; 1.5ml/kg+LPS) control cheese and cheese activated only by LPS
refrigerated at 4 ± 1°C and tested on different days 0, 9 and
Figure 1 shows the variation in the firmness of treated and 18.
untreated cheeses stored at 4±1°C. It can be seen that the The control cheese initially had 3.0 log CFU/g, while the
average firmness value of the cheeses decreases directly LPS-only activated cheese showed a slight decrease to 2.93
following over time. log CFU/g.
In fact, the firmness values of control cheeses fell from 0.28 The reduction in this flora in cheeses made from milk
to 0.2 N after 18 days of refrigerated storage. preserved by activation of the LPS system suggests that
For cheeses activated only by LPS, firmness fell from 0.277 activation of this system in the milk before cheese
to 0.21 N after 18 days of refrigerated storage. manufacture could be of great practical importance (Seifu et
The incorporation of the essential oil with LPS had no al., 2004) [26].
significant effect on firmness compared with the controls Moreover, Seifu et al, (2004) [26] found that preservation of
and those activated by LPS. cheese milk by the LPS system can be used to improve the
microbiological quality and flavour of cheese.
Microbiological quality At t9 and t18, there was a significant difference in all the
The results of the evolution of the microbial flora of the samples. Moreover, the two other combinations resulted in a
cheeses at (t0) and after 9 and 18 days of storage at 4±1°C. significant reduction in mesophilic aerobes at t0.

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There was a total absence of mesophilic aerobes for the LPS However, no significant difference was recorded in the
combination with the highest dose of 1.5ml/kg, which may evolution of yeasts and moulds in the LPS-activated cheese
be due to the high concentration of thyme essential oil compared with the control.
applied. In fact, the addition of thyme essential oil to fresh cheese
The combination of LPS with the highest dose (1.5ml/kg) at had a significant effect on yeasts and moulds for the two
t0 resulted in a significant decrease compared with the doses incorporated (1 and 1.5ml/kg) with LPS, with a more
control cheeses and those activated by LPS and those treated effective effect for the 1.5ml/kg dose at t0 and t18.
with a dose of 0.5ml/kg combined with LPS. Our results are in agreement with Guiraud (2003) [11] who
During the 9th day of storage, we recorded a sudden showed that the incorporation of thyme essential oil at 1ml
increase in the concentration of aerobic mesophilic bacteria /kg and 1.5 ml/kg have a significant effect on the
in the cheese preserved by the addition of 1.5ml/kg of HEt. development of yeasts and moulds.
This can probably be explained by the fact that a chemical
reaction between the proteins and the functional groups of
the essential oils reduced the availability of the active
molecules (Malecky et al., 2007) [15].
From the 18th day, an increase in the number of mesophilic
aerobes was observed in all cheeses. This increase was
significantly less for cheeses activated by LPS combined
with thyme essential oil. We conclude that incorporating
1.5ml/kg of thyme essential oil with LPS into the cheese has
a significant effect on the development of mesophilic Fig 4: Effect of the incorporation of encapsulated thyme essential
aerobes. oil on yeasts and molds in fresh cheese (Control; LPS; 0.5ml/kg
Our result is similar to that of Guiraud (2003) [11] who +LPS; 1 ml/kg +LPS; 1.5ml/kg+LPS)
demonstrated that the incorporation of thyme essential oil at
different concentrations (1 and 1.5ml/kg) showed that only Sensory analysis
at the highest concentration 1.5 ml/kg was there a The sensory analysis, hedonic test, was carried out directly
significant effect on the inhibition of the development of after treatment at t0, t9 and t18 days of storage at 4°C on
mesophilic aerobes. control cheeses, activated by LPS and cheeses with different
This highlights the importance of encapsulating thyme concentrations of encapsulated essential oils (0.5, 1 and
essential oil and combining it with LPS, since a significant 1.5ml/kg) combined with LPS. We chose to carry out these
reduction in the development of mesophilic aerobes was analyses throughout the 18-day storage period on the basis
observed at different essential oil concentrations (0.5, 1 and of the cheese quality assessed.
1.5 ml/kg) with LPS. The characterisation of the sensory properties of the cheeses
was analysed using the radar diagram constructed with the
scores obtained for the different parameters evaluated by the
panellists.
On the basis of the deterioration index reported, if the
overall acceptability score was less than 5, the product
would be considered a putrid food (Lainez et al., 2008) [13].
The highest score for aroma was given to cheeses with an
essential oil concentration of 0.5 ml/kg with LPS, with an
average of 5.58 out of 9 at t0, which decreased over time to
an average of 5.25 at t9 and an average of 4.33 at t18. On the
Fig 3: Effect of encapsulated thyme essential oil incorporation on other hand, the lowest score was awarded to the cheese with
total mesophilic aerobes of fresh cheese (Control; LPS; 0.5ml/kg the highest concentration of essential oil in combination
+LPS; 1 ml/kg +LPS; 1.5ml/kg+LPS) with LPS, which totalled 4.75 out of 9 and fell over time to
3.75 at t18.
Yeasts and moulds For the other descriptors, such as salty taste and acid taste,
Yeasts and moulds in cheese are considered to be spoilage the highest scores were attributed to the cheeses with the
organisms leading to flavour and texture deterioration, lowest concentration of essential oil combined with LPS,
including softening and discolouration (Aly et al., 2012). which scored 5, 33 and 5.25 out of 9 respectively at t0 and
For yeasts and moulds, the trend was similar to that for decreased over time to values of 4.58 and 3.58 over 18 days
mesophilic aerobes. of storage.
There was a significant reduction in the microbial load For the other descriptors, neither colour nor odour had a
following the combination of encapsulated essential oil at significant effect on all the cheese samples. On the other
different concentrations with LPS at t0. hand, time had a significant effect throughout the shelf life.
In fact, the lowest concentration (0.5ml/kg) and the average For the descriptor "texture", the score attributed by the panel
concentration (1ml/kg) combined with LPS showed average with all the concentrations combined with LPS did not have
yeast and mould values of 1.8 and 1.37 log CFU/g a significant effect (p>0.05) compared with the control and
respectively, compared with the control, which was initially the cheese activated only by LPS. This is in agreement with
at 2.34 log CFU/g and 2.31 log CFU/g for those only our result found in the paragraph studying the rheology of
activated by LPS. The levels were even lower with the OEt the cheeses, in which we found that the incorporation of
concentration of 1.5 ml/kg combined with LPS (0.85 log essential oils with LPS had no significant effect on the
CFU/g). texture of the cheeses.

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National Journal of Advanced Research www.multidisciplinaryarticle.in

However, the panel found that the texture deteriorated overall acceptability at t18, whereas there was no significant
significantly throughout the shelf life. Similarly for overall difference between t0 and t9. On the other hand, overall
acceptability, there was no significant difference between all acceptability was strongly correlated with colour, odour and
the samples. However, shelf life had a significant effect on texture (p<0.01).

Fig 5: Effect of incorporating encapsulated thyme essential oil on the organoleptic properties of fresh cheese

Effect of hurdle technologies on the evolution of the the We were able to demonstrate that the storage combination
shelf life of cheese had no significant effect on texture throughout the duration
Estimation of the use-by date using the accelerated aging of refrigerated storage. On the other hand, these
test combination had a significant impactt on all the physico-
The spoilage kinetics of a foodstuff is a representation of the chemical parameters (pH, titratable acidity, total dry extract,
deterioration of a parameter A as a function of time and fat, defatted dry extract and moisture) throughout the shelf
temperature. These kinetics are generally of order zero or of life.
order one. On the other hand, the panel did not detect any significant
The determination of the order of the spoilage reaction is effect on the descriptors (colour, odour, texture and overall
obtained by comparing the coefficient of determination R2 acceptability) for the two combinations throughout the shelf
of the linear regression of the three kinetic models related to life.
a quality criterion A by drawing the graphs In addition, the determination of the shelf life showed that
Order zero: A = f(t) the use of these combination resulted in its extension of by
Order 1: Ln (A) = f(t) 3.63 days, compared with 3.09 days for the LPS cheese
Order 2: (1/H) = f(t) alone. On the other hand, the combination of thyme
Determination of the use-by date by monitoring the essential oil encapsulated with LPS gave different results
evolution of yeasts and molds,indeed the microbial limit of depending on the concentration. The dose of 0.5 ml/kg with
acceptability was estimated by fitting the experimental data activation of the lactoperoxidase system resulted in a shelf
to the Gompertz equation modified by Corbo (Zantar et al., life of 4.19 days, compared with 8.01 and 10.40 days
2013) [29] respectively for 1 and 1.5 ml/kg with LPS.
A concentration ≥ 105 CFU / g of yeasts and molds marks
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