The Pharma Innovation Journal 2023; 12(2): 2514-2521
ISSN (E): 2277-7695
ISSN (P): 2349-8242
NAAS Rating: 5.23
TPI 2023; 12(2): 2514-2521
© 2023 TPI
www.thepharmajournal.com
Received: 14-12-2022
Accepted: 19-01-2023
Kavita K Solanki
Jaipur Zila Dugdh Utpadak
Sahakari Sangh Ltd, Jaipur,
Rajasthan, India
Bikash C Ghosh
Dairy Technology Division,
ICAR- National Dairy Research
Institute, Bengaluru, Karnataka,
India
Arvind Kumawat
Jaipur Zila Dugdh Utpadak
Sahakari Sangh Ltd, Jaipur,
Rajasthan, India
Corresponding Author:
Kavita K Solanki
Jaipur Zila Dugdh Utpadak
Sahakari Sangh Ltd, Jaipur,
Rajasthan, India
Shelf-life study of fortified sweetened milk kefir during
storage
Kavita K Solanki, Bikash C Ghosh and Arvind Kumawat
Abstract
Shelf-life study of fortified sweetened milk kefir with stabilizer (WS) was done randomly after every 3
days interval and compared with fortified sweetened milk kefir without stabilizer (WOS) and control
kefir. All of three were analysed for physico-chemical, sensorial, textural properties and coliform counts
during storage. After 6thday of storage acidity of fortified sweetened milk kefirs was significantly
(p<0.05) higher than control kefir till 15th day of storage. For water holding capacity, it was increased up
to 6th day of storage and then decreased towards the end of storage for all the three kefirs. Flavour of all
the three kefirs were decreased significantly (p<0.05) after 6th day of storage. No significant (p>0.05)
difference was found between the fortified sweetened milk kefir WS and WOS till 9th day of storage, but
significant (p<0.05) difference was there between them on 12th day. As the storage progressed colour and
appearance scores became significantly (p<0.05) higher for fortified sweetened milk kefir than control
kefir, but did not differ significantly (p<0.05) between fortified sweetened milk kefir WS & WOS. A
lower decrease in body and texture of fortified sweetened milk kefirs than control kefir was found during
the storage. On 12th day of storage, the overall acceptability scores were significantly (p<0.05) lower for
control and fortified sweetened milk kefir WOS as compared to fortified sweetened milk kefir WS. All
the textural properties were highest for fortified sweetened milk kefir added with stabilizer (WS).
Coliform counts found to be absent in all the fresh samples and no coliforms growth was observed till the
end of storage.
Keywords: Shelf life, fortified sweetened milk kefir, textural properties, sensorial properties
Introduction
Fermented dairy products are extensively consumed owing to their health benefits and
refreshing effects. It is considered that their popularity is due to the efficient use of consumer-
driven flavours (Jensen and Kroger, 2000) [17]. The known ethnic fermented products in India
are such as Dahi (curd), Mishti Doi (sweetened curd), Shrikhand, Lassi and Chhach or Mohi
(buttermilk), Chhurpi, Somar, Philu and Shyow. (Dewan and Tamang, 2007) [5]. Kefir is one of
the fermented milk products which is very new to India but thought to be consumed in
Caucasus Mountains and Mangolia (Duncan, 1986 [6]; Libudzisz & Piatkiewicz, 1990 [23];
Dzwolak & Ziajka, 2000) [7] for thousands of years. As the kefir has acidic and yeasty taste, it
can be relished less by consumer. Sweetening of kefir will make the taste milder; improve
flavor and overall acceptability which will be more relished by Indian population.So, in one of
our studies, we (Solanki and Ghosh, 2021) [27] reported the optimization process of fortified
sweetened milk using kefir grains with sugar (6, 8 and 10%), fibres (inulin, oat and soya, each
3%) and stabilizer (0.1% pectin) and concluded that an acceptable quality of fortified
sweetened milk kefir can be prepared with 0.1% pectin, 6% sugar and 3% inulin. In addition to
this we also characterized fortified sweetened milk kefir with stabilizer (WS) and compared
with fortified sweetened milk kefir without stabilizer (WOS) and control kefir (Solanki and
Ghosh, 2021) [27]. Further in order to know the changes in chemical and biochemical
composition of the characterized product in due course of storage and find maximum time of
the product to be consumed safely, the current study was designed to evaluate the physico-
chemical, sensorial, textural properties and coliform counts of fortified sweetened milk kefir
with stabilizer (WS) during storage and compared it with fortified sweetened milk kefir
without stabilizer (WOS) and control kefir.
Materials and method
Fresh whole milk was procured from the Experimental Dairy Plant of ICAR- National Dairy
Research Institute, Bengaluru.
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Kefir grains were obtained from U.S. Food grade High
methoxy pectin from HiMedia Laboratories Pvt. Ltd.,
Maharashtra.Refined crystalline cane sugar was purchased
from local market, Bengaluru, India. Dietary fibres used in the
study viz. soya fibre (Fimbrim®), oat fibre (Vitacel® HF-
600®) and inulin were supplied from M/s Solae Company
(U.S), M/s J. Rettenmair and SohneGmbh (Germanay) and
M/s DKSH India Pvt.. Ltd. (Bangalore, India), respectively.
Cups made up of polypropylene material were used for
packaging of milk kefir.
Preparation of fortified sweetened milk kefir
Devlopment and optimization process of fortified sweetened
milk kefir (WS) has been reported by Kavita and Ghosh,
(2021) [27] in detail. Briefly whole fresh milk was standardized
to 3% fat and homogenisation was done. Fortified sweetened
milk kefir (WS) was prepared with the addition of stabilizer
i.e. pectin at 0.1% level, sugar at 6% level and 3% inulin on
milk basis. Heat treatment up to 90-92°C for 10 minutes was
done followed by cooling at 30°C. At this temperature kefir
grains were inoculated at 4 g/L to the milk and incubated at
30ºC for about 20-24 h in B.O.D incubator till the titratable
acidity reached at 1% lactic acid. The fermented product was
thoroughly stirred and sieved through nylon sieve of 1/20-
inch mesh size to retrieve the kefir grains. Finally product was
filled in polypropylene cups and kept for maturation for 24 h
at refrigerated temperature (6-8°C) and stored. For comparison
control kefir was prepared in same manner as the optimized
product without addition of pectin, sugar and inulin. Kefir
without stabilizer (WOS) was also prepared with 6% sugar
and 3% inulin on milk basis.
Shelf-life evaluation of developed product
The fortified sweetened milk kefir (WS & WOS) and control
kefir were packed in 100 gm PP cups (thickness – 183 µm)
with lids and stored in triplicates at 6-8°C. Samples were
drawn randomly after every 3 days interval and were analysed
for physico-chemical properties (pH, acidity and water
holding capacity), sensorial properties (flavour, colour and
appearance, body and texture and overall acceptability),
textural properties (firmness, consistency, index of viscosity
and stickiness) and coliform counts.
Physico-chemical analysis
PH
The combined glass electrode was calibrated against standard
buffer of pH 4.0, pH 7.0and pH 9.2 at 30°C. The pH of
fortified sweetened milk kefir samples was measured directly
by inserting the electrode into the sample. The reading was
recorded.
Titratable acidity
The acidity of fortified sweetened milk kefir was measured as
per the method of AOAC947.05 (2012) for milk. The sample
was well mixed, and 10 g of sample was taken in a beaker.
Phenolphthalein indicator of 2-3 drops was added and titrated
against 0.1 N NaOH till the first appearance of faint pink
colour. The acidity was expressed as % lactic acid by weight.
Water holding capacity
The water holding capacity of the kefir samples was
determined using the previously described method by Harte et
al. (2003) [15] with slight modifications. About 40 g kefir
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sample was placed in a 50 ml centrifuge tube and weighed.
Tube was centrifuged at 6000 rpm at room temperature for 15
minutes. The separated whey was pipetted out and weighed.
The following formula was used to calculate WHC (%).
W2 − W1
WHC (%) = × 100
W2
Where: W1 = Weight of whey (g)
W2 = Weight of kefir sample (g)
Sensory evaluation
Sensorial assessment of fortified sweetened milk kefir was
carried out by an expert panel of minimum five judges. The
sensory panel was selected based on the normal sensitivity for
basic colour/appearance, body/texture and flavour of
sweetened milk kefir. Sensory panellists were well trained on
evaluation of sweetened milk kefir and were made familiar
with the test methods used. Duo-trio tests were used to
determine a candidate’s ability to detect differences among
similar products with different ingredients.
Product temperature was about 6-8°C and test assessment was
carried out at room temperature. Prior to assessment, each
type of sweetened milk kefir which is packed in
polypropylene cups was cooled and coded with alphabets.
The evaluation was carried out under proper lighting.
Panellists were asked to judge the kefir sample on 9-point
hedonic scale (9=like extremely; 1=dislike extremely). The
judged parameters were flavour; body and texture; colour and
appearance; overall acceptability. Water was provided for
mouth washing between evaluations of samples.
Textural analysis
Textural characteristics such as firmness, consistency, index
of viscosity and stickiness were determined using TA-XT plus
Texture Analyser (Stable Microsystem, UK) with P/25
cylindrical probe. For determining the textural characteristics,
the fortified sweetened milk kefir sample of 200 ml was taken
in 250 ml beaker and assessment was carried out at 6-8°C.
The probe travelled at a speed of 1.0 mm/s up to 10 mm
distance into the kefir sample from the surface and then
returned to the original position generating force-time curve.
The positive peak of the curve gave firmness (Newton), the
negative peak gave stickiness (Newton), the area of positive
peak gave consistency (Newton-second) and the area of
negative peak gave index of viscosity (Newton-second).
Enumeration of coliform count of fortified sweetened milk
kefir
Reagents
a) Violet Red Bile Agar (VRBA): VRBA of 41.5 g was
dissolved in 1000 ml of distilled water. The content was
boiled to dissolve the medium completely. Sterilization
was done in autoclave at 15 psi (121°C) for 15 minutes.
b) Tri sodium citrate (2%): Tri sodium citrate of 20 g was
dissolved in 1000ml of water and autoclaved at 15 psi
(121°C) for 15 minutes.
Procedure
Enumeration of Coliform count in fortified sweetened milk
kefir was performed during storage study at 3 days interval.
Product sample (11 ml) was diluted in 99 ml of sterile 2%
(w/v) tri-sodium citrate. Appropriate dilutions 100 and 101
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were pour-plated. Coliform counts were enumerated on
VRBA and incubated at 37 °C for 24 h. Plates containing
colonies with dark red coloration (if any) were enumerated
and recorded as log CFU/ml of the product.
Statistical analysis
The results obtained in the present study were subjected to
analysis of variance (ANOVA) using IBM SPSS statistics 23
software. Data obtained from the triplicate trials were used for
statistical analysis.
Result and discussion
Shelf-life studies of fortified sweetened milk kefir
The shelf life is the time during which the product remains
wholesome and exhibits no physical or organoleptic defect. It
is the recommended maximum time of the product to be
consumed safely under expected (or specified) conditions of
packaging, distribution and storage.Visible yeast growth was
observed on the surface of control and fortified sweetened
milk kefirs on 14th and 17th day of storage respectively. Hence
the sensory analysis was carried out till 12th day and other
analysis were carried out till 15th day of storage. The
observations are as follows:
K (WOS) – kefir without stabilizer, K (WS) – kefir with stabilizer
Fig 1: Changes in acidity (a) and pH (b) of control and fortified sweetened milk kefirs during storage
The initial higher acidity (p<0.05) in fortified sweetened milk
kefir as compared to control may be due to the utilization of
inulin by bacteria present in kefir grains (Rodrigo et al., 2014)
[25]
that continue to increase till 6th day in control kefir and 9th
day in fortified sweetened kefirs. Acidity and pH of fortified
sweetened milk kefir with stabilizer were not differ
significantly (p<0.05) across the day because of stabilizing
effect of pectin. The decrease in acidity and corresponding
increase in pH in kefir may be attributed to enhanced yeast
growth as the storage progressed (Güzel-Seydim, 2000 [14],
Collar, 1996) [4]. Grønnevik et al. (2011) [11] also found that
in initial days of storage of kefir, lactic acid bacteria
increased, whereas yeast levels increased later days of
storage.
Water holding capacity: Changes in water holding capacity
(WHC) of all the three kefirs during storage were studied and
shown in Fig. 2. The water holding capacity was significantly
(p<0.05) higher for both fortified sweetened milk kefirs than
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Changes in physico-chemical properties of fortified
sweetened milk kefir during storage
Acidity and pH
The acidity of control and fortified sweetened milk kefirs
were significantly (p<0.5) increased till 6th day and 9th day of
storage, respectively and thereafter it significantly (p<0.5)
decreased till 12th and 15th day, respectively (Fig. 1a).
Similarly, pH of control and fortified sweetened kefirs were
significantly (p<0.5) decreased till 6th day and 9th day of
storage, respectively and thereafter it significantly (p<0.5)
increased till 12th and 15th day, respectively (Fig. 1b). Though
initial acidity of fortified sweetened milk kefirs was
significantly (p<0.5) higher than the control, the changes
became non-significant (p<0.5) as storage progressed till 6th
day. Initial pH of 4.47 in control kefir decreased to 4.37 on 6th
day of storage and thereafter it increased to 4.46 on 12th day
of storage. After 6th day of storage acidity of fortified
sweetened milk kefirs was significantly (p<0.5) higher than
control kefir till 15th day of storage. Among pH of all the
three kefirs no significant (p<0.5) difference was observed as
the storage progressed. Similar changes in acidity and pH of
kefir have been reported previously by Irigoyen et al. (2003)
[30]
and Gul et al. (2015) [16].
control kefir. As the storage progressed, WHC was increased
up to 6th day of storage and then it was decreased towards the
end of storage for all the three kefirs.
Fortified sweetened milk kefir (WS) showed increased WHC
which may be due to addition of inulin and pectin. Bot et al.,
(2004) reported that inulin forms network with medium
leading to increased gel strength. Guven et al. (2005) [13]
reported that inulin significantly (p<0.05) increased whey
separation in set-type low-fat yogurt. However, it was
reported that upon storage for 3 days inulin resulted in
decreased whey separation. Everett et al. (2005) [9] reported
that pectin addition to the yogurt improves whey exudation
because of the adsorption of pectin on the surface of the
casein micelles, which results increased water holding
capacity of fermented product. The presence of sufficient
pectin molecules may prevent the formation of large
aggregates or an unstable gel and thereby reduced the extent
of whey separation.
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K (WOS) – kefir without stabilizer, K(WS) – kefir with stabilizer

Fig 2: Changes in water holding capacity of control and fortified sweetened milk kefirs during storage

Changes in sensory attributes of fortified sweetened milk
kefir during storage
Flavour: The changes in flavour of the control and both
fortified sweetened milk kefirs during storage period is given
in Fig 3. The initial flavour scores for both the fortified
sweetened milk kefir were significantly (p<0.05) higher than
the control kefir and the same trend continued till the end of
the storage. Between the fortified sweetened milk kefir,
stabilizer added kefir acquired significantly (p<0.05) higher
flavour score than kefir without stabilizer on 3rd and 6th day of
storage. On 12th day of storage, flavour of control kefir
declined significantly (p<0.05). Staleness, yeasty and tangy
flavour led to the rejection of control kefir. Pronounced yeasty
flavour was detectable in both fortified sweetened kefirs on
15th day of storage, so after that they had been rejected.
Flavour of all the three kefirs were decreased significantly
(p<0.05) after 6th day of storage. In general, as the storage
time passes flavour score of all the three kefirs declined that is
also in agreement with Kilic et al. (1999) [18] who found that
all the sensory attributes of kefir decreased significantly
(p<0.05) with the time and should be eaten within 3 days of
manufacture. The decrease in flavour of kefir might be due to
increased acidity giving acidic taste and fermented odour
(Irigoyen et al., 2005) [16] which was observed during
investigation (Fig 3). A superior flavour score of fortified
sweetened milk kefir was observed than the control kefir
owing to masking of acidic taste with the sweetness of sugar
and inulin. Fortified sweetened milk kefir with stabilizer had
more superior flavour score that might be due to stabilizing
effect of pectin on acidity. Based on the sensory results the
suggested shelf life of control kefir was 12 days while shelf
life of fortified sweetened kefir was 15 days that is also in
agreement with who reported 14 days shelf life of kefir
produced from natural kefir grains.

K (WOS) – kefir without stabilizer, K(WS) – kefir with stabilizer

Fig 3: Changes in Flavour of control and fortified sweetened milk kefirs during storage

Body and texture: Fig. 4 depicts the changes in body and were more in fortified sweetened milk kefirs than control
texture of kefirs during storage. The body and texture scores kefir throughout the storage. However, no significant
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(p<0.05) difference was found between the fortified 6th day of storage body and texture score was increased in
sweetened milk kefir with stabilizer and without stabilizer till both the fortified sweetened milk kefirs but after 6th day it was
9th day of storage, but significant (p<0.05) difference was decreased but the difference was non-significant (p<0.05) in
there between them on 12th day. As the storage progressed, till fortified sweetened milk kefir with stabilizer.

K (WOS) – kefir without stabilizer, K(WS) – kefir with stabilizer

Fig 4: Changes in body and texture of control and fortified sweetened milk kefirs during storage

A lower decrease in body and texture of fortified sweetened
milk kefirs than control kefir during the storage period might
be attributed to the stabilizing effect of inulin and pectin that
created a gelling effect to the fortified sweetened milk kefirs
and improve body and texture. (Parker et al., 1994 [24]. A
decrease in body and texture of all the three kefirs is closely
related with the findings of Tarakci and Kucukoner (2003) [29]
and Salwa et al. (2003) [26], they reported a decrease in score
of body and texture of yogurt during storage. Moreover, there
was lower whey separation in fortified sweetened milk kefir
that resulted in the improvement in body and texture.
Colour and appearance: Changes in colour and appearance
of all the three kefirs during storage were studied and shown
in Fig. 5. The initial colour and appearance scores of all the
three kefirs were not significantly (p>0.05) different, but as
the storage progressed scores became significantly (p<0.05)
higher for fortified sweetened milk kefir than control kefir,
however scores did not differ significantly (p>0.05) between
fortified sweetened milk kefir with stabilizer and without
stabilizer. Across the day difference in colour and appearance
scores was non-significant (p>0.05) for both fortified
sweetened milk kefirs but significant (p<0.05) difference was
there for control kefir after 6th day of storage. Improved
colour and appearance of fortified sweetened milk kefir may
be attributed to inulin and pectin that produces glossy
appearance in the product. But, visible yeast growth, presence
of whey pockets and air bubbles in control and fortified
sweetened milk kefir on 14th and 17thday of storage
respectively made the products liable for rejection and were
not served for sensory evaluation.

K (WOS) – kefir without stabilizer, K (WS) – kefir with stabilizer

Fig 5: Changes in colour and appearance of control and fortified sweetened milk kefirs during storage

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Overall acceptability
The overall acceptability scores of both the fortified
sweetened milk kefirs were higher than control kefir initially
as well as towards the end of storage period (Fig. 6). Fortified
sweetened milk kefir with stabilizer showed significantly
(p<0.05) higher overall acceptability scores than without
K (WOS) – kefir without stabilizer, K (WS) – kefir with stabilizer
Fig 6: Changes in overall acceptability of control and fortified sweetened milk kefirs during storage
The higher acceptability score in fortified sweetened milk
kefir might be attributed to masking of acidic taste, improved
body and texture as well as bright appearance. But latterly,
decrease in scores may be due to the presence of whey
pockets and gas bubbles towards the end of the storage.
Control samples were rejected due to yeast growth on the 14th
day but no yeast growth was there in fortified sweetened milk
kefir till the 16th day of storage. However, sensory evaluation
for all the three kefirs was limited to till 12th day of storage.
Similarly, Esteller et al. (2006) [8] reported that kefir has shelf
life of 13-15 days because of the organic acids formation.
Changes in textural properties of fortified sweetened milk
kefir during storage
Changes in textural properties of all the kefir samples during
storage are shown in Fig 7. It was observed that firmness of
both the fortified sweetened milk kefirs was significantly
(p<0.05) higher than control kefir and the same trend
continued throughout the storage. Firmness of all the three
kefirs changed significantly (p<0.05) with time. Firmness of
control and both the fortified sweetened milk kefirs were
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stabilizer. On 12th day of storage, the scores were significantly
(p<0.05) lower for control and fortified sweetened milk kefir
without stabilizer but for fortified sweetened milk kefir with
stabilizer no significant (p>0.05) difference was observed till
12th day of storage.
increased till 3rd and 6th day of storage and thereafter
decreased. The higher firmness in fortified sweetened milk
kefir as compared to control may be attributed to the water
holding capacity of inulin microcrystals and pectin (Franck,
2002 [10]) as evident from Fig. 7.
Consistency was also significantly (p<0.05) higher for both
fortified sweetened milk kefir than control kefir throughout
the storage. Improved consistency in fortified sweetened milk
kefir than control kefir may be attributed to the enhanced
exopolysaccharide (EPS) production by kefir grains in the
presence of stimulating effect of added inulin. Similarly,
higher values of stickiness and index of viscosity in inulin
incorporated sweetened kefir indicate viscous nature of the
product as inulin gels produces richer texture (Leporanta,
2001 [22]). All the textural properties were significantly
(p<0.05) higher for the fortified sweetened milk kefir added
with pectin. It is in agreement with Syrbe et al. (1998) [28],
they stated that aggregation and settling of casein particles
during storage results in whey separation but the addition of
high methoxyl pectin is known to overcome this defect.
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K (WOS) – kefir without stabilizer, K (WS) – kefir with stabilizer
Fig 7: Changes in textural properties of control and fortified sweetened milk kefirs during storage
Evaluation of coliform counts in fortified sweetened milk
kefir during storage
In the present study, coliform counts were checked for all the
three kefir samples (control, fortified sweetened kefir without
stabilizer and fortified sweetened kefir with stabilizer) and
found to be absent in all the fresh samples and no coliforms
growth was observed till the end of storage. This indicates
that proper hygienic measures were taken during the
production of the kefir samples.
Conclusion
Fortified sweetened kefir (WS) showed better physico-
chemical, sensorial and textural properties as compared to
control kefir. Due to yeast and tangy flavour control kefir was
rejected on 12th day of storage but fortified sweetened kefir
(WS) can be consumed safely up to 15th day of storage. All
textural properties were highest for fortified sweetened kefir
added with stabilizer (WS) during storage render it safely
consumed for longer period.
Acknowledgments
The first author acknowledges the receipt of fellowship from
the institute of ICAR-National Dairy Research Institute (SRS)
to carry out the research.
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