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Growth of Lactic Acid Bacteria In Probiotic Purple Cheese

From Carabao’s Milk


Jocelyn G. Daclag1 and Roberta D. Lauzon2 Journal of Science, Engineering and Technology
Vol. 3:113- 125(2015)

Abstract

“Kesong puti,” a local variety of soft cheese, is known in some parts of the
Philippines but it is less acceptable in the Visayan region. The
incorporation of sweetened purple yam is seen to improve the cheese’s
flavor and overall appeal. The addition of probiotic starter curter was
intended to produce a probiotic flavored cheese with viable number of live
microorganisms. This study was conducted to evaluate the growth of live
lactic acid bacteria (LAB) on the flavored cheese. It also determined the
formulation and processing condition that produced sufficient level of live
microorganisms on the probiotic product to deliver its intended effects.
Results of the study revealed the microbial count to be generally high,
exceeding 106 cfu/g, in the treatments incubated at 44°C for 15 hours and
treatments with low concentrations of salt. The incubation period showed a
direct relationship with the microbial count. Growth of LAB increased with
the corresponding increase of the incubation period. Salt, however, offered
an inverse relationship with the microbial count. At constant incubation
time, treatment with 1.0% salt concentration produced higher bacterial
growth than treatments with 2.5% and 4.0% salt.

Keywords: purple yam (Dioscorea alata L.), response surface


methodology, contour plots , Streptococcus thermophilus,
Lactobacillus delbrueckii subsp. bulgaricus

1.0 Introduction portion of the milk, commonly


referred to as curd. Enzymes and/or
The “kesong puti”, also acid are added to milk and coagulate
called as white cheese or native the casein proteins and fat
cheese, is a semi-soft cheese often component (Brown 2011). The
made from fresh carabao’s milk. It is production of “kesong puti” in the
processed by either curing milk with Philippines is an old cottage industry
rennet or coagulation by vinegar in Laguna, Bulacan and Cebu
(Claudio et al., 2014). Cheese is provinces (Claudio et al., 2014). The
basically made from the solid product is popular in these
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provinces. However, the bland taste digestive tract to re-establish


of “kesong puti” reduced its themselves. Probiotics play
acceptability in most part of the important roles in mitigating illness
Visayas region. The addition of and preventing infections (FAO
sweetened purple yam into the 2006; Weichert, Schroten and Adam
traditional “kesong puti” was made 2012). They promote good health by
in this study to improve the flavor, improving the balance of the
sensory appeal and marketability of intestinal microflora and mucosal
the product. Purple yam (Dioscorea defense against pathogens (Karimi et
alata L.), locally known as ubi, al., 2012). Thus, it is believed that
contains fair amounts of nutrients the addition of live microorganisms
and anthocyanin pigment which into the flavored native cheese will
promote health. It has appealing increase the product’s aesthetic and
purple color and a desirable flavor. nutritional appeal.
Probiotic purple cheese is a vinegar- The applications of probiotic
coagulated semi-soft cheese flavored bacteria in food which are mostly
with purple yam. It is made associated with yogurt, kefir and
following the basic process of cultured drinks (Karimi et al. 2012)
“kesong puti” production. It is also have extended into the development
added with live microorganisms, of probitic cheese. Some research
Streptococcus thermophilus and efforts have been devoted to
Lactobacillus delbrueckii subsp. developing probiotic hard or semi-
bulgaricus, using a commercial hard cheese. A probiotic cheese
yogurt starter culture. containing Lactobacillus rhamnosus
Probiotics is defined as “live and Lactobacillus acidophilus was
microorganisms administered in manufactured by Mills DA in
adequate amounts that confer a Norway. The product was used in a
beneficial effect on the host” (FAO clinical trial to improve the intestinal
2006). The popularity of probiotic microbiota and fecal immune marker
food products in the market has of some elderly volunteers (Lahtinen
consistently increased. Probiotic et al., 2010). Gomes and Malcata
bacterial cultures are associated with (1998) also developed a probiotic
the restoration of the healthy balance semi-hard cheese from goat milk.
of the gut flora and resistance to The Department of
infection. They are intended to assist Microbiology of Tartu University
the body’s naturally occurring flora developed an original probiotic semi
and enable good bacteria within the -soft cheese. It was prepared basing
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on the Estonian open-texture, smear- environment in the gastrointestinal


ripened, semisoft “Pikantne” cheese tract, creating an environment that
(Songisepp et al., 2004). Karimi et favors the survival of the
al. (2012) also cited several microorganisms (Karimi et al.,
researchers who developed various 2011; Karimi et al. 2012). Karimi et
probiotic cheeses with different al. (2011) have cited three major
probiotic strains. These probiotic areas that affect the stability of the
products include Minas fresh cheese, probiotics in cheese. These include
fresh cream cheese with inulin the formulation factors, process
supplement, cottage cheese, cheddar factors, and packaging materials and
cheese, cheddar-like cheese, soft systems. Process factors cover the
cheese, Petit Suisse cheese, strain of probiotic bacteria and
Argentinean fresco, Festivo cheese, microbial interactions, pH and
Canestrato Pugliese hard cheese, titratable acidity of the product,
Iranian white-brined cheese, Turkish hydrogen peroxide, molecular
white cheese and several others. It is oxygen, growth promoters and food
evident that fewer efforts have additives, salt, microencapsulation,
focused on making probiotic and ripening of the cheese. The
flavored soft cheese. Probiotic process factors, on the other hand,
purple cheese or “kesong ubi” is a include incubation temperature, heat
potential product to offer a healthier treatment, types of inoculation, and
variation of the traditional “kesong storage temperature. This
puti” that is known in the information implied that different
Philippines. cheese formulation and process may
The cheese has been reported also have a different effect on the
to have a good potential than the survival and stability of the bacteria
fermented milk for delivery of in the probiotic product.
probiotic microorganisms into the Researches on probiotic
intestine for its chemical and cheese have included different
physical characteristics. It has higher strains of probiotic bacteria
pH value and lower titratable incorporated into different types of
acidity, high buffering capacity, cheese. Gomes and Malcata (1998)
greater fat content, high nutrient utilized Bifidobacterium lactis and
availability, lower oxygen content, Lactobacillus acidophilus to
and denser matrix of the texture produce the probiotic semi-hard goat
(Karimi et al., 2011). It creates a cheese. Songisepp et al., (2004) used
good buffer against the high acidic the Lactobacillus fermentum strain
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Vol. 3:113- 125(2015)

ME-3 with the “Pikantne” cheese. It also determined the effect of the
The probiotic cheese that was formulation and process of cheese
produced had antimicrobial and on the growth of LAB on the
antioxidative properties. Hard finished product.
cheese, soft cheese, cottage cheese,
white cheese, white-brined cheese, 2.0 Methodology
fresh cream cheese and other
cheeses have been subject to studies. The study used the 33
Viability of the probiotic bacteria fractional factorial experiments
and other important product following the Central Composite
attributes has been evaluated in Design (CCD). A total of 15
these different cheeses (Karimi et al. treatments were used with
2011). However, none of the concentrations of salt (1, 2.5, 5%, w/
mentioned cheese types included w), levels of sweetened ubi (30, 40,
flavored semi-soft cheese. The 50%, w/w), and incubation period
incorporation of purple yam may (5, 10, 15 hours) as the main
alter the cheese formulation and variables (Table 1).
processing conditions. It may also Percentage of the ingredients
affect the possible viability of used in the formulation of the purple
probiotic bacteria in the product. cheese was based on the volume of
The challenge in this study is the the fresh milk used in the study. The
need for the probiotic bacteria to quantities of vinegar and probiotic
grow and maintain its sufficient medium (starter) were held constant
number during the process and for all treatments. The probiotic
storage of purple cheese to deliver purple cheese was processed using
its intended effect on the the flow shown in Figure 1. A liter
gastrointestinal tract. Hence, this of fresh carabao’s milk for each
study is conducted. treatment was pasteurized and
This study focused on the immediately cooled to lukewarm
evaluation of the growth of lactic temperature. Vinegar (5% of the
acid bacteria (LAB) on the purple milk’s volume) was added for
cheese considering the adjustments curdling. Whey was drained while
in the formulation and process the curd was added with salt as
variables. The growth performance determined on the variation for the
of LAB was assessed as indicated by treatments. Sweetened ubi at varying
the microbial count on the processed levels for the different treatments
purple cheese from carabao’s milk. was added into the mixture. The
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Table 1. The different treatments of the central composite design (CCD) for
33 fractional factorial experiments used in evaluating the probiotic
purple cheese.

probiotic medium (1.5% of the of LAB. Samples were diluted in


milk’s volume) was added after the buffered dilution water and plated in
mixture was thoroughly blended. triplicate using pour plating method.
The said probiotic medium was The plates were incubated at 370C
obtained from a commercial plain for 24 to 48 hours. Counting of the
yogurt and activated in a pasteurized total plate count (TPC) and
fresh carabao’s milk. Activation of enumerating of the colony forming
the culture was done using the units (cfu/g) was done to determine
yogurt maker. The cheese was then the microbial load of the product.
molded to its shape and incubated at The colony appearance of the LAB
constant temperature for varying isolated from the products was
time as required in the treatments. evaluated and recorded. Gram-
The 15 treatments of the probiotic staining method was also performed
purple cheese were subjected to to examine the morphological
microbial evaluation to examine the characteristics of the
growth of lactic acid bacteria on the microorganisms and to verify if they
finished product. The microbial are typical characteristics of LAB.
count of the LAB in the product was The data for the microbial
determined using a GPY-BCP agar, count of the products were subjected
a specific medium for the cultivation to Response Surface Regression
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(RSReg) to determine the effects of variables on the response studied.


independent variables on the The data were also subjected to
microbial quality of the product. Analysis of Variance (ANOVA) to
Response surface plots were made determine the effect of the
for each run condition to clarify the regression models on the microbial
different effects of the factor count of the finished product.

RAW MILK

Pasteurizing

Cooling

Curdling

Whey draining

Salting (1, 2.5, 4%)

Adding of sweetened ubi (30, 40, 50%)

Blending

Inoculating of probiotic medium

Molding/pressing

Incubating at 44°C (5, 10, 15 hours)

Storage

PROBIOTIC PURPLE CHEESE


Figure 1. Process flow for the production
of probiotic purple cheese
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3.0 Results and Discussion by Karimi et al. 2012). According to


Xu, Boylston and Glatz (2006), the
The microbial counts of the bacterial counts should exceed the
different treatments of probiotic suggested minimum count of 107
purple cheese are summarized in cfu/mL or cfu/g. It is the
Table 2. The count (cfu/g) of the recommended level to affect the gut
LAB ranged from 2.9 x 104 to 2.04 x environment and provide health
108 with ten treatments (T1, T2, T3, benefits. Seven of the 15 treatments
T6, T7, T8, T9, T10, T11 and T13) for the purple cheese had reached
obtaining counts over 106. The food the count of 107 cfu/g.
industry has generally applied the It was observed that
recommended level of 106 cfu/g at microbial count was generally high
the time of consumption for (exceeding 107) at treatments which
probiotics bacteria. This bacterial were incubated at 44°C for 15 hours
concentration is considered because and treatments with low
it is cost-effective and concentrations of salt. The majority
technologically attainable rather of the health benefits related with
than to achieving a specific health probiotic bacteria depend on cell
effect in humans (Roy 2001 as cited viability. Thus, it is essential to

Table 2. Summary of the mean* microbial count of probiotic purple cheese.

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maintain high levels of viable cultured in GPY-BCP agar produced


probiotic cells in foods. The small to medium-sized, round/
treatments which exceeded the circular, convex and moist colonies.
minimum count for probiotic food, Majority of the colonies were white,
therefore, qualified the requirement some cream colored and a few were
for probiotic products. Cheese is a yellowish. A clear zone of inhibition
good vehicle for the delivery of (Fig. 2a) was observed surrounding
probiotics into the intestines as it in most of the colonies. These
provides several advantages over cultural characteristics are typical
other fermented milk products characteristics of Streptococcus
(Karimi et al. 2012). thermophilus and Lactobacillus
Figure 2 presents the colony delbrueckii subsp. bulgaricus, the
appearance of the microorganisms microorganisms in yogurt which
isolated from the probiotic purple was used as the probiotic medium
cheese. The different samples for the product.

Figure 2. Cultural characteristics of microbial isolates from the


probiotic purple cheese.

Result of the gram staining bulgaricus.


showed a gram-positive culture The linear surface plots of
which is typical of lactic acid the microbial count as affected by
bacteria, especially that coming from the different formulation and
yoghurt. The spherical or cocci processing variables are shown in
shaped culture which were formed in Figures 3a, 3b and 3c. Each variable
chain confirmed the presence of the was held at constant level, consistent
S. thermophilus bacteria. Another to the levels used as the pivot in
culture that is also gram positive rod generating contour and surface plots
-shaped is an indication of the L. while the effect of the other
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Figure 3. Surface plots of microbial count (CFU/g) of probiotic “kesong


ubi” at constant: (a) 15 hours incubation period; (b) 2.5% salt;
and (c) 40% sweetened ubi.

variables were evaluated. As the 2.5% (Fig. 3b). Microbial count also
incubation period was held constant, exhibits a direct relationship with
results showed (Fig. 3a) that the incubation period when level of
microbial count is inversely related sweetened ubi was held constant
to the levels of salt. Microbial count (Fig. 3c). The result implies that
decreased with the corresponding increasing the incubation period
increased of salt levels. This result provided a chance for the increase in
implies that salt had an inhibitory growth of LAB in the product. The
effect on the growth of lactic acid same trend was observed by Xu et
bacteria. The result can be expected al., (2006) who reported that the
because lactic acid bacteria are not microbial counts of all fermented
halophilic in nature. milk products increased significantly
On the other hand, microbial when incubated at 45°C and stored
count of probiotic purple cheese at 4°C for one day. The conditions
showed a direct relationship with the resulted to the microbial counts of
incubation period while salt 1.74 x 107 to 1.62 x 109 cfu/mL.
concentration was held constant at Analysis of variance for the
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microbial count of probiotic “kesong interaction action of incubation


ubi” (Tables 3 and 4) showed that all period and salt concentrations
regression models have highly suggested that high levels of these
significant (p<0.01) effects on the variables will reduce the survival
microbial count of the cheese. rate of the microorganisms in the
Linear and quadratic models had a finished product. However, the
lump effect on the microbial count positive relationship of the
of the probiotic cheese. The incubation period when salt was
interaction of salt and incubation held at constant level (Fig. 3b)
period have a significant negative elaborated that the salt contributed
effect on the microbial count at more of the detrimental effect on the
p< 0.01. This negative effect of the growth of LAB.

Table 3. Analysis of variance of the different regression models for


the microbial count of the probiotic purple cheese.

Table 4. Parameter estimates for response surface of microbial count of


probiotic purple cheese from carabao’s milk.

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The effect of the incubation and at treatments with low


period can be related to the growth concentrations of salt. The
curve (growth rate and growth treatments which exceeded the
characteristics) of LAB. Microbial minimum recommended level for
growth curve have four main stages: probiotic food qualified the
(a) lag phase, microbial population requirement for probiotic products.
does not change; (b) exponential The growth of LAB on the probiotic
(logarithmic) phase, microbial cells products may be affected by the
grow exponentially; (c) stationary levels of salt, sweetened ubi and the
phase, a few cells and a few cells incubation period. The linear and
multiply due to nutrient shortage and quadratic models of the variables
accumulation of waste products; contributed a lump effect on the
living population is stable; and (d) growth of the LAB on the product
death (decline) phase, rate of cell while the cross interaction of the salt
death is higher than the rate of level and incubation period bring a
multiplication (Ray 2004). With the significant effect on the viability of
incubation time of 5 to 15 hours, the microbial count. The microbial
microbial count increases as count is inversely related to the salt
incubation time was increased. It concentrations. It decreases with the
may be the stage in which the corresponding increase in levels of
growth of bacteria is at the the salt. Microbial count is also
logarithmic phase. This fact will directly related with the incubation
also explain the negative effect of period from 5 to 15 hours. It means
the incubation period during the that increasing incubation period
preliminary studies. It is possible may provide a chance for the growth
that at some time of incubation of LAB in the product to increase up
beyond 15 hours, the bacterial to its viable level to effect in the
growth had reached the stationary gastrointestinal tract.
and death phases.

5.0 References Cited


4.0 Conclusion
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The microbial count of the Foods: Preparation and
probiotic purple cheese was Principles, Philippine
generally higher at treatments which Edition. Philippines:
were incubated at 44°C for 15 hours Cengage Learning Asia Pte
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Vol. 3:113-125(2015)

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