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Evaluating Physical and Biological Methods of Preservation of Traditionally

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RESEARCH ARTICLE

Evaluating Physical and Biological Methods of Preservation of Traditionally Produced

Ethiopian Cottage Cheese (ayib) on their Keeping Quality
Zerihun Asafa1 and Esayas Abreha2
1,2
Ethiopian Institute of Agricultural Research, Holeta Agricultural research center, Food Science and Nutrition Research.
Po box 031, Holetta, Ethiopia

AR T IC L E I NFO AB S T RA C T

Received: December 12, 2021 Ayib is an Ethiopian acid and heat coagulated milk product that is typically
Revised: January 21, 2022 made in the home. Their shelf life is limited, and their organoleptic properties
Accepted: February 10, 2022 are inconsistent. Using bio-protective lactic acid bacteria and physical drying
procedures, this study aimed to improve the keeping quality, organoleptic
Key words:
property, and consistency of ayib. In this work, two drying procedures (air oven
Bio-protective
dry and microwave oven) were used to evaluate the keeping quality, proximate
Dehydration
composition, physicochemical property, organoleptic consistency, and
Ayib
microbiological safety of two bio-protective L. rhamnosus and L. paracase. The
Organoleptic.
protein, fat, and crude ash contents of microwave dried and oven-dried ayib
were 25.67 and 23.85 percent, 1.94 and 1.91 percent, and 2.03 and 2.14 percent,
respectively. The physicochemical property pH exhibits a broad reduction
during the first 12 days of storage, with minor increases later. The treatable
acidities of biologically treated ayib increased somewhat for the first 6 hours
and thereafter decreased marginally. These dehydrated ayib samples, on the
other hand, rose modestly after 6 days of storage. During storage, the water
activities of dehydrated ayib rose linearly. Both biologically and dehydrated
ayib's organoleptic properties and sensory shelf life deteriorated during storage.
Dehydrated ayib had an 18-day sensory shelf life, while biologically treated
ayib had a 9-day sensory shelf life when stored at room temperature. To
summarize, both biological and dehydration procedures were found to be
effective in improving the keeping quality and organoleptic consistency of
*Corresponding Address: conventionally processed ayib. Particularly in poor countries' rural milk
zafuase@gmail.com, processors, where there is little market access and no chilling facilities to
+251911552657 preserve perishable dairy products.

Cite This Article as: Asafa Z, Abreha E, 2021. Evaluating physical and biological methods of preservation of
traditionally produced ethiopian cottage cheese (ayib) on their keeping quality. Inter J Appl Sci Engr, 9(1): 31-37.
www.ijapscengr.com

INTRODUCTION 2006). Ayib is an Ethiopian cottage cheese created from

Cottage cheese is created from the milk of various
mammals and is produced industrially using traditional
processing methods in practically every country on the
globe. There are over 2000 different types of cottage cheese
available. Traditionally, Ethiopians fermented and
consumed a considerable percentage of their milk each
year. Ergo (traditionally fermented milk), ayib (Ethiopian
cottage cheese), Kibe (traditional butter), and Arera are
common fermented milk products prepared in Ethiopia
using traditional processing methods (buttermilk). The
most popular dairy products consumed in the country are
ergo and ayib, which are fermented goods (Ashenafi,
traditionally fermented milk by churning out the fat and
slowly heating the defatted milk (buttermilk) until a
discrete curd mass forms and floats over the whey. It is a
well-known popular milk product that is extensively
consumed by many ethnic groups in various parts of the
country, and it is one of Ethiopia's most popular homemade
fermented milk products manufactured using traditional
methods (Gonfa et al., 2001; Tesfaye et al., 2011a). It is a
major source of nutrients and is a mainstay of the diet. It
can be eaten raw as a side dish or flavored with strong
spices, salt, and another herb (Gonfa et al., 2001).
The safety of milk and milk products is a major
concern in underdeveloped countries since they are

31
 32
produced in unsanitary settings (Ashenafi, 2002; Tesfaye et
al., 2011b). The initial load of microorganisms in the raw
milk themselves, as well as post-processing, might result in
a high load of microbes in the product and low maintaining
quality in the conventional unsanitary method of ayib
manufacturing (Andualem and Geremew, 2014).
According to Mehari and Ashenafi (1995), mesophilic
aerobic bacteria, yeasts, and enterococci were found in ayib
samples gathered from an open market in the Hawassa
district of Ethiopia, with 108, 107, and 107 cfu/g,
respectively. Bacillus cereus and Staphylococcus aureus
had 102-103 cfu/g.
In ayib samples obtained from the Jimma zone of
Ethiopia, Tekletsadik and Tsige (2011) found 6.13, 5.96,
5.84, and 5.5 log cfu/g of aerobic mesophilic bacteria,
Enterobacteriaceae, yeasts, and mold, respectively. The
counts of aerobic mesophilic bacteria, coliforms, and
molds in homemade ayib samples taken from five North
Showa regions of Ethiopia were 5 to 8 log cfu/g, 2.4 to 4.8
log cfu/g, and 3.1 to 4 log cfu/g, respectively, according to
Mamo et al., (2016).
These high microbial loads are a major source of food-
borne diseases and the primary cause of product
deterioration or spoilage. As a result, the shelf life of
traditionally manufactured ayib was only 2-3 days when
stored at room temperature. They lose their
physicochemical and organoleptic qualities and can no
longer be consumed by humans.
Some LAB has been approved to produce metabolites
such as organic acids (lactic and acetic acid), hydrogen
peroxide, ethanol, diacetyl, acetaldehyde, and other low-
molecular mass antibacterial chemicals like bacteriocins
(Vuyst, 2004). The use of these LAB with bio-protective
properties has been shown to extend the shelf life of items
by inhibiting food deterioration and harmful bacteria, while
also providing a health benefit to the user at a reasonable
cost (Gomes and Malcata, 1998).
Water activity, on the other hand, was a prevalent
component in the shelf life/spoilage of foods. The oldest
method of extending the shelf life of food was to reduce the
water activity of the meal by dehydrating it. However, the
practice of dehydrating food has an impact on the
nutritional constituents of foods, particularly protein and
vitamin content. So, the goal of this study was to increase
the shelf life and safety of ayib by employing bio-protective
LAB and physical dehydration to reduce the moisture
content and water activity of the products to levels below
the optimal for spoiling and pathogenic microbe
development.
MATERIALS AND METHODS
Study Area
This study was done in Holeta dairy research
Laboratory/Holeta microbial biotechnology research
Laboratory.
Preparation of ayib
Ayib was prepared as the method described by Tesfaye
et al., (2011c). In detail, raw cow milk of thirty-two liters
was fermented spontaneously at an ambient temperature of
20-240C for 48 hours in a stainless-steel milk can. The
fermented milk was defatted by agitating with an electrical
Inter J Appl Sci Engr, 2021, 9(1): 31-37.
churner for 30 minutes. The floating fat was removed with
a sterile spoon. Defatted milk (buttermilk) was distinctly
heated at 50°C in a water bath for 55 minutes until a
separate curd mass was formed. The curd (ayib) was
separated from the whey and after cooling the curd solid
mass at room temperature, they were filtered through sterile
cheesecloth.
Physical Methods of Drying
In this method, a hot air-drying oven at 50oC and
Microwave dehydration at 350W was used until the
moisture content is equivalent to 10-18%.
Oven Drying
Ayib samples of 800 grams were uniformly spread on
a tray dryer having aluminum sheets and dried at 50oC of
the air-dry oven for 4-6 hours until a constant weight was
obtained following the method described by (Pinho et al.,
2017).
Microwave Dehydration
Ayib samples of 800 grams were dried in a microwave
at 350 W and the mass was registered every 15 seconds
until a constant weight was obtained.
Biological Method of Treatments
Candidate Lactic Acid Bacterial Isolates
Four lactic acid bacteria L. rhamnosus (LB-15), and L.
paracasei (LB-7), isolates were used. These isolates were
previously isolated from traditional fermented ergo
samples collected from different agro-ecologies of Ethiopia
and screened and characterized based on their antimicrobial
effects, fermentation capabilities, and flavor
enhancements.
Standardization of LAB Isolates
Each LAB species was cultured on MRS agar
aerobically at 37oC for 48 hrs. The culture concentration
was standardized to 108cfu/ml using 0.5MacFarland
solution. One liter of whey was pasteurized at 80 oC for 30
minutes and cooled to 30 oC at room temperature and the
standardized isolates were mixed in one-liter sterile whey.
Treatment of ayib with the LAB Isolates
Ayib of 400 g was wrapped in sterile cheesecloth and
dipped into pasteurized whey inoculated with standardized
LAB cultures for 30 minutes and re-drained for 1 hr.
Organoleptic Properties of Physical and Biologically
Treated Ayib
Ayib products treated with each LAB isolates and
physically dried ayib was soaked in whey heated at 84oC
for 30 minutes and re-drain for 10 minutes. One control
(Ayib) prepared similarly and not treated) was provided for
the sensory test. The sample was randomized and coded
with three-digit figures and served at 18-24 oC in a dish
separately. A test form comprising five sensory attributes
was given to each assessor. The sensory assessments were
conducted every three-day interval for up to 15 days of
storage. Ten semi-trained panelists of adult males and
female panelists were involved in the test. A glass of water
was provided for each panelist and, taught to rinse with
water while working with the samples (Yang & Li, 2010).
 33
The panelists have given written instructions and
evaluated the products for color, flavor, odor, texture, and
overall acceptability using a five-point hedonic scale: 1 =
Dislike Extremely, 2= Dislike Moderately, 3= Neither like nor
a dislike, 4= Like Moderately, and 5= Like Extremely. In the
assessment of the organoleptic characteristics of the product,
one was equal to the worst and five was equal to the best.
Proximate Composition
Protein Content Determination
The macro Kjeldahl method as described by (AOAC,
2006) was used to determine crude protein content. From
each sample, two gram was introduced into the digestion
flask. Ten grams of copper sulfate and sodium sulfate in the
ratio of 5:1 and 25 ml of concentrated sulphuric acid was
added to the digestion flask. The flask was placed in a
digestion block in a fume cupboard and heated until
frothing ceased given a clear and light blue coloration. The
mixture was allowed to cool and diluted with distilled water
until it reaches 25 ml of volumetric flask. Then, 10 ml of
the mixture was poured into the distillation apparatus and
10 ml of 40% sodium hydroxide was added. The released
ammonia by boric acid was allowed to continue until 10 ml
of boric acid is treated with 0.02 M of hydrochloric acid and
the green color changed to purple. Finally, the percentage of
nitrogen in the sample was determined and calculated as the
method described by Eshetu and Asresie, (2019a).
Fat Content Determination
The soxhlet solvent extraction method as described by
(AOAC, 2006) was used to determine the fat content. Two
grams of the sample were weighed in a flask of known
weight with the extractor mounted on it. The thimble was
held halfway into the extractor the weighed sample was
carefully transferred into the thimble, and the thimble was
plugged with cotton wool. The extraction was carried out
at the temperature of 40 – 60 oC for 8 hrs. The solvent was
removed by evaporation and then, the remaining part in the
flask was dried in the oven at 80 oC for 30 minutes and then
cooled in desiccators. The flask was reweighed and the
percentage fat was calculated as follows
Determination of Total Solid Contents
The total solids were determined as described by (AOAC,
2006). A sample of 3 g was weighed into a dry crucible of
known weight. The sample was then dried in an air-drying
oven at 100 ± 2 °C until a constant weight was achieved
between consecutive measurements at 30-minute intervals.
The total solid of the sample was calculated as follows:
Where: W = Weight of the crucible, W1 = Weight of
crucible plus sample, W2 = Weight of oven-dried crucible
plus sample.
Inter J Appl Sci Engr, 2021, 9(1): 31-37.
Determination of Ash Contents
The ash content was determined by the direct heating
method as indicated in (AOAC, 2006). In this method, 3 g
each of the samples was measured into a crucible of known
weight, the sample was burned to ash in a muffle furnace
for 5 ± 1 hrs. at 550 oC. It was then cooled in a desiccator
and the percent of ash was finally determined as follows
Where: W1 = Weight of crucible, W = Weight of sample
test portion, W2 = Weight of dish and dry sample.
pH Measurement
The pH of each biologically and physically treated
ayib sample was determined using a digital pH meter (pH
meter 704, Herisau, Switzerland) after calibrating it using
standard buffers of pH 4.00 and 7.00. Ten grams of ayib
was mixed with 10ml of distilled water having their pH
standardized to 7.00. The pH probe was immersed in a
solution and a pH reading was taken.
Titratable Acidity
The titratable acidity of the products was analyzed
following the methods of (International Organization
Standard 11869, 2012). Ten milliliters of the mixture was
titrated with 0.1N standard NaOH with four drops of
phenolphthalein indicator until faint pink color is obtained.
Determination of Water Activity
The water activity values of the products were
determined using a water activity meter (Aqua Lab 4TE,
USA) according to the guidelines of the manufacturer and
procedures as described by ((Mathlouthi, 2001; Bawa,
2020) at 25°C.
Sensory Shelf-Life Estimation
Ayib products treated with different bio-protective
LAB starter cultures and ayib dehydrated with air dry oven
and microwave oven were stored at an ambient temperature
of 18-24oC. The sensory test was done every three days
intervals until 15 days. During the sensory test, dehydrated
ayib were warped with sterile cheese close and soaked in
hot whey of 84 oC for 30 minutes, and drained for 10
minutes. The overall acceptability cut-off point was 3.5, as
was indicated by (Garitta et al., 2015). The rate constant k-
value of the products was predicted using overall
acceptability versus storage time linear regression and first-
order kinetic reaction. The sensory overall acceptability of
each product was changed to natural log, and the linear
regression equation of natural log of sensory overall
acceptability versus storage time was drowning to get rate
constant (K). Using rate constant K, the initial overall
sensory satisfaction (A0) and quality limit (cut-off point)
(At) at five points hedonic scale, the shelf life of the
product was calculated following Arrhenius first order
reaction equation as described by (Tsironi et al., 2009).
 34
Where: Ao = Natural log of initial (1st day overall sensory
mean score), At = quality limit (Cut off point = 3.5), K=
rate constant
Microbiological Safety Analysis of the Products
Common indicator microbes of a total aerobic
mesophilic bacterial count, total coliform, and yeast and
molds count were done following standard methods of
(International Organization Standard 4833-1, 2013) for total
aerobic mesophilic bacteria, (4832, 2006) for a coliform
count and (ISO 21527-1) for yeast and mold count.
Experimental Design
The experimental design is CRD with triplicate
Statistical Analysis
Statistical analysis of the treatments was made using
SAS 9.4 version 2014. The normality of data was tested
using a Q-Q probability plot and analysis of variance
(ANOVA) was done at α=0.05. The mean difference of
proximate composition of treatments was tested using
turkey’s tests at α = 0.05 levels of significance.
RESULTS AND DISCUSSION
The result of the proximate composition analysis of
each ayib treatment was summarized in Table 1. The
proximate composition of the products ranged from 20.09
to 20.96% for total solid, 15.26 to 15.40% for protein, 2.01
to 2.09% for fat, and 1.15 to 1.30% for ash content in
biologically treated ayib. These proximate compositions
were no significant difference at α<0.05 between treatment
and the control sample. This indicates that these LAB
isolates were no impact on the proximate composition of
the products during storage/ preservation of products.
During food, preservation maintaining the nutritional
quality as well as the safety and organoleptic
properties/stability of the products are critical to attracting
consumers. The finding in this study was related to Eshetu
and Asresie, (2019b) who found 21.29 to 21.67%, 14.53-
16.78%, 1.15 to 1.17% for total solids, protein, and ash
content respectively in traditionally prepared ayib products
collected from east Gojam zone. The fat content in this
study was relatively higher than these found by Eshetu and
Asresie, (2019b) this might be due to the variability in
breed, feed of the animals, or lactation period of a cow from
which the raw milk was obtained (Patil and Patil, 2020;
Dandare et al., 2014).
Concerning the nutritional analysis of the dehydrated
ayib, particularly total solid, protein, and fat content there
is a significant difference(P<0.05) between air dry oven
drying at 52oC and microwave dried ayib at 750 W. Total
solid content of 96.16 and 94.15% was observed in micro-
oven-dried and air-dry oven respectively. Significantly, (p
< 0.05) higher total solids, protein, and fat content were
observed in microwave dried ayib products Table 2. The
proximate composition was 25.67 and 23.85% for protein,
1.94, 1.91% for fat, and 2.03, and 2.14% for crude ash in
the microwave and oven-dried ayib respectively. Bawa,
(2020b) reported that the fat content of microwave dried
crickets was relatively higher fat content than oven drying.
The variation in proximate composition between the two
methods might be due to differences in the drying
Inter J Appl Sci Engr, 2021, 9(1): 31-37.
techniques drying temperature and time. Microwave drying
was a small time of exposure to heat which maintains the
nutritional value of the overnight hot air-dried oven.
(Bawa, 2020; Bawa, 2020).
The crude ash content of oven-dried and microwave-
dried ayib were no significant difference (p<0.05) between
the two methods of dehydration. Crude ash is the total
mineral content of the product which is not destroyed by
thermal treatment due to its low volatility compared to
other food nutrients (Bawa, 2020).
The physicochemical properties of pH, titratable
acidity, and water activity of these treatments were changed
during the storage of the product at an ambient temperature
of 25oC. The result of this study showed that the pH of ayib
products treated with bio-protective LAB was steeply
decreased during the first ten days of storage and later they
were moderately increased as showed in Figure 1a. The
decrease in pH might be due to the acidification of the
sugar, most commonly lactose, which is fermented to lactic
acid by bio-protective lactic acid bacteria. However later
they were increased moderately this might be due to the
interaction of microbial metabolites in the products some
microbes use lactic acid as a substrate. The pH of the
dehydrated ayib product had not significantly changed
during the storage time. This might be due to the low
moisture content and water activity of the products, there is
no or low microbial activity and physicochemical change
of the products. These treatments were good keeping
quality for the product.
The total titratable acidity of ayib product treated with
bio-protective LAB was increased for the first six days and
later they were declined linearly this might be due to the
exploitation of substrate lactose in the products. These
dehydrated ayib products were slow increment of total
titratable acidity, this might be associated with the slow
growth of lactose fermenting microbiota in the products
associated with low moisture and water activity content of
the products.
The water activity of ayib products treated with bio-
protective LAB was shown to slow increment during
storage of the products.
Whereas, these dehydrated ayib products were steadily
increasing during storage of the products at an ambient
temperature of 25oC this might be due to the hygroscopic
nature of the products.
The sensory overall acceptability of the products during
the storage was shown in Figure 2. The sensory overall
acceptability of each product was decreased as the storage
time increased. Relatively higher overall acceptability was
observed by treatment AYO1 and lower overall acceptability
was observed in a control sample. The dehydrated ayib were
relatively better sensory overall acceptability as compared
to biologically treated products. This is correlated with the
low physicochemical change of products due to lower
moisture content as well as water activity.
As the storage time increases the physicochemical
properties of the product were changed which affects the
organoleptic properties and consumers' acceptability of the
products (Wartu, 2018).
Ayib provides a suitable environment for the growth
of many microorganisms, especially mesophilic bacteria,
yeasts, and molds at room temperature due to high water
activity and protein levels.
 35 Inter J Appl Sci Engr, 2021, 9(1): 31-37.

Table 1: Proximate composition of ayib treated with bioprotective lactic acid bacteria
Treatments Total solid Protein Fat Ash
AYBG-15 20.78±0.1 a 15.41±0.02 a 2.05±0.01a 1.19±0.01a
AYSB-7 20.96±0.24 a 15.26±0.31 a 2.09±0.06a 1.15±0.06a
ab a a
Cont 20.09±0.12 15.40±0.17 2.01±0.03 1.30±0.10ab
Where AYBG-15= L. rhamnosus, AYSB-7= L. paracasei, and Cont = control ayib sample without any treatment

Table 2: Proximate composition of dehydrated ayib products

Treatments Total solid Protein Fat Ash
AYM1 96.16±0.91a 25.67±0.50a 1.94±0.06a 2.03±0.06a
AYO1 94.15±0.90b 23.85±0.56b 1.91±0.05b 2.14±0.04a
Where AYM1= ayib dried using a microwave oven, AYO1= ayib dried using an air-dry oven

Table 3: Microbial quality of biological and physically treated ayib products

Treat TPC Coliform Yeast &Mold
AYBG-15 1.96±0.02b 0.19±0.05b 0.35±0.03c
AYSB-7 1.90±0.02b 0.15±0.09b 0.19±0.10d
AYM1 0.12±0.06d 0±00c 0.56±0.07b
c c
AYO1 0.15±0.12 0±000 0.14±0.13d
Cont 2.16±0.02a 1.43±0.06a 1.30±0.02a
Where: AYBG-15 and AYSB-33 were LAB treated products and AYM1 = microwave oven dried product, and AYO1= air-dry oven-
dried ayib products

Table 4: Sensory shelf life of treated ayib products

Product Quality criteria Storage temp Quality limit R2 Estimated shelf life (in days)
AYBG-15 Overall acceptance 20-25oC 3.5 0.98 8
AYSB-33 3.5 0.99 9
AYM1 3.5 0.96 18
AYO1 3.5 0.98 16
Cont 3.5 0.82 4
Where: AYBG-15 and AYSB-33 were LAB treated products AYM1 = microwave oven dried product and AYO1= air-dry oven-dried
ayib products.

4.9
4.75 AYSB-7
4.6 AYG-23
pH value

4.45 AYOV1
4.3 AYMV1
4.15 Cont
Storage time in days
4
1st 6th 12th 15th

Fig. 1a: pH value of the product during storage at 25oC

0.98
Titrable acidity in % m/v

0.94
0.9 AYSB-7
0.86 AYG-23
0.82 AYOV1
0.78 AYMV1
0.74 Storage time in day Cont
0.7
1st 6th 12th 15th

Fig. 1b: Total titratable acidity of the products during storage

0.9 AYBG15
Water activity

0.8 AYSB7
AYM1
0.7
AYO1
0.6
Cont
0.5
1st 6th 12th 15th

Storage time in Days

Fig. 1c: Water activity of the products during storage time of the products
 36
Fig. 4: Overall sensory acceptability of the products during storage
The sensory shelf life of the product was shown in
Table 3. Relatively higher shelf life of 16 and 18 days was
observed in oven dry and microwave dried ayib products
stored in an open-air of room temperature respectively. In
this study, these bio-protective probiotic LAB treated ayib
products were 8-9 days of sensory shelf life at ambient
temperature storage.
All the treatments were relatively more than two times
than the control samples (4 days).
Conclusions
In this study physical treatment of microwave and
oven drying methods were better shelf-life improvement of
16 and 18 days at room temperature storage. These two
treatments AYM1 and AYO1 were also better consumer
preference. So that these treatments were better for
improving the keeping quality of cottage cheese in rural
areas where there are no cooling facilities.
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