International Journal of Advanced Biotechnology and Research (IJBR)
ISSN 0976-2612, Online ISSN 2278–599X,
Vol-7, Special Issue3-April, 2016, pp510-517
http://www.bipublication.com
Research Article
Study of biochemical and organoleptic characteristics of acidic alcoholic
fermentation milk (kefir) as a dietary food for people suffering from lactose
intolerance
Mohadeseh Nemati2,3, Mahshid Mohammadian2,3, Elmira Roshani Asl2,3,
Zafar Gholinejad3, Attabak Toofani Millani3, Mehrdad Vakili4,
Shiva Mehran5 and Mahsa Nemati1*
1
Department of food sciences & technology,
Facultyof Agriculture, Urmia University, Urmia, Iran
2
Student Research Committee,
UrmiaUniversity Of Medical Sciences, Urmia. Iran
3
Department of Biochemistry, School of Medicine,
Urmia University of Medical Sciences, Urmia, Iran
4
Department of food sciences & technology,
faculty of agriculture, ZanjanUniversity, Zanjan, Iran
5
Department of Biochemistry, Rab,eRashidi University, Tabriz ,Iran
Corresponding author: Mahsa Nemati,
ABSTRACT
The fermented milk, kefir is one of the oldest products originating Caucasus Mountains. This Fermentation product
has high biological values .Kefir products due to lactic acid bacteria that produce the enzyme lactase could be a
suggestive food for people suffering from lactose intolerance. Today, consumers want high-quality food with minimal
processing steps to produce healthy product with minimal levels of preservatives can kept in an acceptable time. Our
Research were to produce kefir on an industrial scale with high quality with optimal biochemical and organoleptic
characteristics that could be a suitable diet in lactose intolerant patients. In this research, milk, whey, kefir starter, mint
essence was used to generate product. produced Kefir in accordance with mixture- amount( 5 variables )including the
certain concentration of each compounds( milk (100,75,50, 25,0) and the whey (100, 75, 50, 25,0), the mint essence
(200, 125, 50), the starter (4, 2.5, 1)) and storage time (14, 7, 0) were examined.Samples chemical (PH, acidity state)
and biological and organoleptic properties were analyzed. The resultsshowed with increase in preserving
time,microbialpopulationcount decreasedbut PH increased. Increasingamount ofessence leadto decreased in PH.
Change in milk, whey concentration effect on PH.but in this case results don’t reached significant level.Other
parameters had no significant change during examination.Overall ourfindings indicated the properties of our
kefirproduct have optimal features according to chemical and organoleptic analysis Also we suggested that kefir could
be considered as suitable diet for patients with lactose intolerance.
Key words: kefir, lactose intolerance.
INTRODUCTION
Milk industry and its products have played a
major role in nutrition and health at all stages of
human lives. The fermented milk, kefir is one of
the oldest products originating from Caucasus
Mountains residents that have produced it.(1-5)
This Fermentative product has high biological
value and high levels of the amino acids such as
cysteine , which is concerned as a valuable and
good quality food for human consumption.(5-8).
Probiotics are living microorganisms (in most
cases, concluded bacteria) that are similar to
beneficial microorganisms found in the human
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
gut, and they also are called Bacteria Friendly or
good bacteria. When we consume probiotics we
actually add bacteria to our body. (9-12)the main
reason for the use of probiotics is to maintain
human health. Kefir is a consumption probiotic
which traditionally has been prepared and is often
flavored with fruits and is the most known alcohol
acid fermentation milk that in addition to the
traditional production, importance of this dairy
products recognized from 1980s.during this time
until now extensive research's has been made on
the optimization of industrial production and the
increasing diversity of this product. Kefir
properties compounds include lactic acid, carbon
dioxide, ethyl alcohol and aromatic compounds
that made during fermentation. (13-16) Milk
lactose intolerance is a common problem among
Asian and African descent. Kefir products due to
bearing lactic acid bacteria that produce the
enzyme lactase is an appropriate diet for people
suffering from lactose intolerance. Indeed Bacteria
and yeast in kefir, increases the secretion of
digestive enzymes to better and faster food
digestion. In fact kefir is suggestive for people
suffering from lactose intolerance, who cannot
digest large amounts of lactose. (17-21)
Kefir digests yeast cells and has beneficial effect
on the intestinal flora. Since kefir is a fermented
product, has almost no lactose amount and is
therefore useful for people with lactose
intolerance. As a result, the amount of lactose in
kefir fermentation process reduced and beta levels
galactosidase increased. Since Proteins are
partially digested, in vivo they are easily
autolysis.(22-24). According to studies the kefir
can reduce many symptoms of lactose intolerance,
including abdominal bloating, diarrhea, abdominal
gas, Kefir is rich in vitamins, minerals, essential
amino acids and proteins that make it as easily
digestible food also kefir is rich in calcium, amino
acids, methionine, folic acid, magnesium,
Phosphorus, copper, and vitamin B12 , vitamin K
that help the in vivo autolysis (spontaneous
oxidation), cell growth and energy supply.(2426)Throughout humanhistory use of plants as a
Mahsa Nemati, et al.
nutritive and medicinal source were common.
Today, consumer'sapplicant is high-quality food
with minimal processing steps to stay longer and
acceptable
time
without
preservatives.
Therebythis demand with strict rules on the use of
synthetic preservatives, hasprovidedfields of
expanding research to find natural substances that
have antioxidant and anti-microbial properties.
The aim of presented study was to Preparation
andinvestigation of kefir on an industrial scale in
order to produce high quality product with optimal
biochemical and organoleptic characteristics with
minimal level of preservatives. This optimized
product could be a suggestive suitable diet for
individuals suffering from lactose intolerance. (25,
26-27)
MATERIALS AND METHODS
Milk Preparation
Pasteurized milk is prepared with 5/2% fat
content from factory then the milk is heated at
temperature of 95 ° C for 5 minutes and then
cooled in temperature of 30 °to 35°C( the
temperature of making starter). After have being
cooling stage is the starter added. That there
starter values were at 3 different amount comprise
(1, 2.5, 4%) with 3 types of starter (CHN-22,Laf 4 and 10ABY- ) . the same values of each starter
types is weighted, and after calculation, the
necessary amount of starter added to milk. After
adding starter stage, the bottles filled with milk
then these bottles placed in 37 ° C incubator (1618 hours) to be put in the fermentation stage , and
after spending fermentation period and reaching
optimal PH, it must be immediately arrived and
reserved in refrigerator temperature (4 ° C). if our
sample has different milk and whey amount ,
whey heating at 65 ° C for 15 minutes and then
cool down to a temperature of 30-35°C, and then
mix to starterized milk, and then incubated with
similar fermented milk samples .after fermentation
process in following cooled.
Preparation of whey
Required whey was prepared from Sahar cheese
factory (Vila cottage cheese), which used
traditional method for cheese producing. Prepared
511
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
whey is containing lactose and other nutrients.
This product should be immediately used after
preparation because long preservation increases its
acidity. after Whey prepare ,heating stage were
performed in a condition of 65 ° C for 15 minutes
and then is cooled up to an optimal temperature of
starterization which was 30-35° C in starter
amounts of 1-2 / 5-4 %(whey starter cofactor
named Laf -4 ABY-10 and CHN-22 ) then
incubated in the incubator at 37 ° for a period of
16 -18 hours then cooled in 4 °C .
Adding Essence
We used the mint Essence, which has been bought
from the Tehran Zard-Band Co. Mint essence is
added to samples in quantities (200, 125,50 ppm).
Then is caped. Adding essence occurs when the
starter bacteria is some acted, and then essence is
added. After incubation at 4 ° C for 10 hours the
sample is cooled in order to prevent the formation
of too much carbon dioxide and froth formation.
Samples Tests and examinations:
PHmeasurement: We regulate the device
according to sample temperature and with buffer
solution 4 and 7 then measure the PH value.
Acidity-measurement
using base solution Our kefir sample aciditymeasurement performed
in this way we mixed 10 ml of kefir sample with
the following 2 drops of alcoholic phenol
PHetalien 1 g % then we add base solution
,whenever base solution cause a purple color via
interaction with kefir and kefir it does not lose its
purple color that means sample were neutralized
with base solution. At this point, read the burette
level and the amount of consuming base
determined.
Acidity Calculation:
Acidity % =
N: ml of consumed base
V: test volume
Microbial culture: Culture medium According to
the recommendation instruction of brochure,
provide and then as before mentioned, we heat it
and then autoclaved. After autoclave (121 °c, 20
min), we place culture medium in Ben-Mary for
45 min.then we provided the ringer and 9 cc of it
were poured. In to each test tube,then autoclaved.
Also we sterile the pipets (180 °cfor 1.5 hours or
UV exposure for 15 min under hood). The
samples transferred to culture medium under
sterile condition. 7 tube with serial dilution
concentration
respectively,-1,-2,-3,-4,-5,-6,-7
prepared. Andone sample of each dilution
cultured. (Inthe sterile plate containing samples
and culture medium then componentsmixed
andincubated in 37°c for 72 hours .after
incubation period microbial growth were seen and
counted.
Sensory evaluation: samples Sensory evaluation
prepared by ten Panelist students from
Biotechnology research center, Urmia university,
Iran, who were carefully selected. To this end, the
evaluation was prepared after subjects drank of
sample then they filled writing form. 9-point
Hedonic test method were used to this evaluation
that carried out on days 1, 7 and 14.
RESULTS AND DISCUSSION
In order to study of biochemical and organoleptic
properties of our kefir product, the tests were
conducted in three steps that investigated the
effects ofsome factors on prepared kefir, these
factors concluded
1)
proportion of milk and whey
2)
the starter percent
3)
essence concentration
4)
preserving time of produced kefir
Acidity: Effect of preserving time and type of
starter on acidityparameter presented in figure1.
Our Findings indicated that higher preserving time in corporates to increase in PH. For example in 14 days
after fermentation the kefir had higher PH than the first week(7 days).there are no significant relationships
between starter type and starter concentrationchange on acidity. Indeed according to our resultsby
Mahsa Nemati, et al.
512
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
increasing starter amount, PHwill not change. As it has been showed in the variance analysis, the final
model is havesignificant level in F-value. And it has significant level in Lack of fit parameter and its
coefficient is 0/9962 and its adjusted coefficient is 0/9876.
Acidity
4.00
D: Starter
3.25
1.37182
1.33491
2.50
1.28653
1.75
1.23814
1.18975
1.14137
1.00
0.00
3.50
7. 00
10. 50
14. 00
E: Tim e
Figure1:the effect of preserving time and type of starter on acidity parameter. Horizontal axis is showing the keeping
time of produced kefir and the vertical axis showstype of starters.
Table1. Variance analysis: It has significant level in Lack of fit and block parameters. the final model is have
significant level in F-value
Source
Block
Model
Linear Mixture
AB
AC
AD
AE
BC
BD
BE
ABC
ABD
ABE
ACD
ACE
ADE
BCD
AC^2
AD^2
AE^2
BC^2
BE^2
ABCD
ABDE
ABC^2
ABD^2
ABE^2
Residual
Lack of fit
Pure Error
Cor Total
Sum of squares
0.32
3.55
2.01
0.14
3.60E-0.3
0.19
0.065
5.51E-0.5
0.016
0.012
8.43E-0.3
1.49E-0.3
0.053
0.035
0.069
1.17E-0.5
4.62E-0.3
0.14
0.047
0.068
2.20E-0.3
4.59E-0.3
6.69E-0.3
0.022
0.02
0.063
0.021
0.014
0.013
1.81E-0.4
3.89
Mahsa Nemati, et al.
df
4
27
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
12
8
4
43
Mean square
0.081
0.13
0.4
0.14
3.60E-0.3
0.19
0.065
5.51E-0.5
0.016
0.012
8.43E-0.3
1.49E-0.4
1.05E-0.3
0.053
0.035
0.069
1.17E-0.5
4.62E-0.3
0.14
0.047
2.20E-0.30.068
4.59E-0.32.20E-0.3
6.69E-0.34.32E-0.3
0.0224.59E-0.3
0.02
0.063
0.021
1.68E-0.3
1.68E-0.3
4.51E-50
Fvalue
116.06
354.11
125.43
3.18
170.82
56.9
0.049
14.3
10.38
7.43
0.13
0.93
46.99
31.28
0.01
4.07
126.46
41.38
59.89
1.94
1.94
4.05
5.9
19.1
17.94
55.37
18.71
p- value prob> F
<0.0001
<0.0001
<0.0001
0.1
<0.0001
<0.0001
0.8292
0.0026
0.0074
0.0184
0.7233
0.3551
<0.0001
0.0001
0.9209
0.0666
<0.0001
<0.0001
<0.0001
0.1893
0.18930.0749
0.0672
0.0318
0.0009
0.0012
<0.0001
0.001
37.21
0.0017
significant
significant
513
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
Effects of the mint essence’s concentration on acidity parameter presented in figure 2.According to finding,
the contourplot shows higher level of essence concentration related to increasing in acidity level.
In higher concentration of essence for example125 ppm toward lower concentrations is more effective on
increase in acidity.
Acidity
4. 00
1.28653
D: Starter
3. 25
1.37182
2. 50
1.33491
1. 75
1.28653
1.23814
1.14137
1.18975
1.14137
1. 00
50. 00
87. 50
125. 00
162. 50
200.00
C : Es senc e C
Figure2: In this contourplot, horizontal axis is showing essence concentration and the vertical axis shows the starter
percent. By increasing the essence concentration the acidity will increased too. The vertical part of plot that is showing
different percent of starter indicates that the acidity will not change by increasing or decreasing of starter
concentration.
The effects of changes in the amounts of milk and whey on the acidity parameter shown in Figure 3.
Two Com ponent Mix
1. 4
Acidity
1. 24
1. 08
0. 92
0. 76
Act ual A 0.000
25. 000
50. 000
75. 000
100. 000
Act ual B100. 000
75. 000
50. 000
25. 000
0.000
Figue3: effects of changes in the amounts of milk and whey on the acidity parameter.
In this contour plot horizontal axisshows the proportion of milk and whey and vertical axis shows the
founded acidity of the tests.
This cantor plot is indicating that by increasing the milk amount up to 70 percent the acidity will increase
and then it will decrease. But by decreasing the amount of whey up to 20 percent the acidity will increase
and then it decrease. So we conclude that the less whey amount, related to higher acidity.
Effects of whey and milk with mint essence concentration on the PH shown in figure4.
Mahsa Nemati, et al.
514
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
pH
200. 000
C
:EssenceC
162. 500
4.28232
4.16294
125. 000
4.04356
3. 80479
3.92418
87. 500
50. 000
Ac t ual B 0. 000
25. 000
50. 000
75. 000
100. 000
Ac t ual A100. 000
75. 000
50. 000
25. 000
0. 000
Figure4: effects of whey and milk with mint essence concentration on the PH. In this contour plot the horizontal axis
shows the different amounts of milk and whey and the vertical axis shows the mint essence concentration.
By increasing the milk amount and decreasing the whey in the produced sample, For example if kefir
produced from pure milk, hashigher acidity level than the sample with 100 percent of whey .indeed in this
case PHchanged but this change did not reached significant level. So we conclude, the changes of milk and
whey proportion don’t have a completelysignificant effect on the PH changes.The contour plot indicated
that the PH will dropped by increasing the essence concentration.
This diagram shows that when the proportion of milk and the mint essence concentration will decreased in
the sample, its PH will be lower than of other samples.
Effects of changes in essence concentration and starter onPH presented in figure5.
pH
4. 00
3.60762
3. 25
D: Starter
3.68116
2. 50
3.7547
3.82824
1. 75
3.90178
1. 00
50.00
87. 50
125. 00
162. 50
200.00
C : Es s enc e C
Figure 5: effects of changes in essence concentration and starter on PH.
In this contour plot, the horizontal axis indicates diagram of mint essence concentration and the vertical
axis indicates different percent of starter. The contour plot means that by increasing essence concentration
the PH will be decreased.
Indeed results means that by increasing essence concentration, the PH will be decreased. This will happen
in such a condition that by increasing starter percent, a significant change will not happen to PH. The more
concentration of mint essence and whey, lead to less PH . For example if the mint essence concentration be
Mahsa Nemati, et al.
515
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
120 ppm and the percent of starter be 2 percent, it has a higher PH in comparison with 200 ppm essence
concentration and 4 percent of starter.
Effects of changes in milk and whey amounts on PH shown in Figure 6.
Two Com ponent Mix
3.79
pH
3.7325
3.675
3.6175
3.56
Act ual A 0. 000
25. 000
50. 000
75. 000
100. 000
Act ual B100. 000
75. 000
50. 000
25. 000
0. 000
Figure 6. the effects of changes in milk and whey amounts on PH.
Based on results as discussed the mutual effect of milk and whey proportion onPHexist. In other words, the
lower percent of PH was observed in the products with lower percent of milk and higher percent of whey.
While the slope of diagram is raising after this 50 percent. This indicates that increasing or decreasing of
milk and whey amounts is effective in the PH percent intactly.
Microbial examination results:
Microbial test results presented in figure7.
One Factor
3. 3E+ 007
Total N
2. 475E+ 007
1. 65E + 007
8. 25E + 006
0
0. 00
3. 50
7. 00
10. 50
14. 00
E: Tim e
Figure7: the microbial count has decreased by increasing the time of fermentation.
Figure of 7 shows that the microbial count has
decreased by increasing the time of fermentation.
Overall our study in order to evaluation of
chemical and bioanalysis of produced kefir
indicated that with increase in preserving time of
product , microbialpopulationcount decreased but
PHincreased. Increasingamount ofessence leadto
Mahsa Nemati, et al.
decreased in PH. Change in milk and whey
concentration affect on PH, but in this case results
don’t reached significant level. Other parameters
had
no
significant
change
during
examination.Overall our findings indicated the
properties of our kefir product have optimal
features according to chemical and organoleptic
516
Study of biochemical and organoleptic characteristics of acidic alcoholic fermentation milk (kefir)
analysis Also we suggested that kefir could be
considered as suitable diet for patients with
lactose intolerance.
REFERENCES:
1. Koushki, M R. Optimization of production
traditional yogurt by industrial method using the
maximum amount of whey Journal of Food Science
and Technology, 2007:4( 2): 28- 19.
2. Koushki, M. R.. Educational book of milk industry
and its products, Shahid Beheshti University.2006
3. Farhudy, F. The nutritional value of milk and its
products, 2004, 61-62.
4. Mir Haider, h. Plants Sciences .plants application in
the prevention and treatment of disease), the first
volume. Publication of Islamic culture. 2000:288295.
5. Hy,R. Waterman, p. Essence of plants (botany,
physiology, chemistry, genetics, biotechnology,
international trade). Translation: Bqalyan, Kambiz.
Andarz Publication.2002:.21- 29.
6. Aridogan‚ B. C.‚ Baydar‚ H.‚ Kaya‚ S.‚ Odemeirci‚
M.‚ Ozbasar‚ D. & Mumcu E. Antimicrobial
activity and chemical composition of some essential
oils. Archives of pharmaced research.2002: 25‚
860- 864.
7. A. Irigoyon‚ I. Arana‚ M. Castiella‚ P. Torre‚ F. C.
Ibanez. Microbiological‚ physicochemical‚ and
sensory characteristics of kefir during storage. Food
Chemistry: 2005: 90‚ 613- 620.
8. Angulo‚ L.‚ Lopez‚E. & Lena Cesar‚ E. Microflora
present in kefir grains of the Galicin Region (northwet of spain). Journal of Dairy Research.1993: 60‚
263- 267.
9. Bayoumi‚ S.. Bacteriostatic effect of some spices
and their utilization in chime- microbilogietechnologie- der- lebensmittel. Thtmanufature of
yoghurt. 1992:14(1/2): 21-26.
10. Chanda Rc. Enhancing market value of milk By
adding cultures. J Dairy Sci‚ 1999‚ 82: 2245- 2256.
11. Holsinger‚ V. H.‚ Posati‚ L. P. & Devilbssi‚ E. D.
Whey beverage: Areview. Journal of Dairy
Science.1984:57(8) ‚ 849-859.
12. Iscan‚ G.‚ Kirimer‚ N.‚ Kurkcuoglu‚ M.‚ Baser‚ K.
H. C. & Demieci‚ F. Antimicrobial screening of
menthe pipertiavessential oils. Journal of
Agriculture and Food Chemistry. 2002:50‚ 39433946.
13. Jelen‚ P The importance of whey components in
food and nutrition. International Dairy Journal.
2003: 13‚ 564- 575.
14. Kissinger‚ J. C.‚ and Zaika‚ L. L.. Effect of majour
spices in Lebanon of food production by starter
culture organisms. Journal Bologna on Acid
Protection. 1987:41(6): 429- 431.
Mahsa Nemati, et al.
15. Kivance‚ M.‚ Akgule‚ A.‚ and Dogna‚ A..
Inhibitory and stimulatory effects of cumin‚
oregano and their essential oil on growth and acid
production of lactobacillus plantarum and
leuconostoc mesenteriodes. International Journal of
Food Microbial. 1991:3(1): 81-85.
16. Kosi Kowski‚ F. V. & Wzorek‚ W. Whey wine
from concentration of reconstituted acid whey
powder. Journal of Dairy Science‚ 1997:60(12) ‚
1982- 1986.
17. Kurman JA‚ Rasic JL‚ Kroger M. En cyclopedia of
fermented fresh milk products an international
inventory of fermented milk cream‚ butter milk‚
whey and relatedproducts. Van Nostrrand Company
(New York) ‚ 1992‚ 368.
18. Maria C. Garcia Fantan‚ Sidonia Martins‚
Inmaculada
Franco‚
Javier
Carballo.
Microbiological and chemical changes during the
manufacturing of kefir made from cow milk‚ using
a commercial starter culture. International Dairy
Journal. 16(2006)‚ 162-167.
19. Maiorella‚ B. L. & Castillo. F.J. (1984). Ethanol‚
biomass and enzyme for whey waste abatement
process. Biochemistry. Aug‚ 157- 161.
20. Mimica- Dukic‚ N.‚ B.‚ Skovic‚ M.‚ Mihaj Lovic ‚
B. & Matavulj‚ M (2003). Antimicrobial and
antioxidant activitics of three menthe spfcies
essential oils. Planta Medica‚ 69‚ 413-419
21. Moshtagi‚ H. & Bonyadian‚ M.. The effects of
some herb’s essential oils s.aureus in feta cheese.
Journal of Medicinal Plants.2007: 6‚ 19- 25‚ 68.
22. Morr‚ C. V. In: J. G.Zadow. Whey and lactose
processing. Elsevier Applied Science.. 1992:5: 135155.
23. Miyamoto‚ T.‚ Morita‚ H.‚ Nishioka‚ K.‚ Kataoka‚
K.‚ Lzumimoto‚ M.‚ & Kuyama T..Constituent
species of lactic acid bacteria from kefir and their
desmutagenic properties. Jap JDairy Food Sci.
199140:111-119
24. Pien‚ J. Use of serum from cheese factories and
lacto in nutrition.Lati‚1943 23‚ 193-199
25. Robinson‚ R. K. The rapeutic properties of
fermented milks. London. Elsevier Applied
Science. 1991:1: 160- 161.
26. Salminen S‚ Ouwehand‚ AC‚ Isolauri E. Clinical
application of probiotic bacteria. Int J Dairy Sci:
1998:8: 563-572.
27. Salwa‚ A.A.‚ Galal‚ E.A.‚ and Neimat‚ A. 2004.
Carrot chemical‚ (microbiocogical properties and
consumer yoghurt: sensory‚ acception. Pakistan
Journal of Nutrition‚ 3(6): 322- 330.
28. Tamime A‚ Robinson R. Yoghurt: Science and
Technology: (2nd Edition) Cambridge‚ uk wood
head publishing‚ 1991.
517