Application of Lactobacillus paracasei spp. Paracasei 1 SKG 44 to Improve

Cheese Quality with Extract of Rampelas (Ficus ampelas Burm F)

I Made Sugitha

, Trisna Damayanti

and Komang Bhanu Artha

Food Science and Technology Study Program, Faculty of Agricultural Technology, Indonesia

Post-graduate Program Udayana University, Bukit Jimbaran, Badung, Indonesia

Keywords: Lactobacillus Paracasei spp. Paracasei 1SKG44, Rampelas Bark (Ficus ampelas Burm F).

Abstract: This research was conducted to find out the application of Lactobacillus paracasei spp. paracasei 1 SKG 44

to improve the quality of cheese by additional of Rampelas Bark (Ficus ampelas Burn F. ) extract as a

coagulant agent. Completely Randomized Design with 5 levels concentration of Lactobacillus paracasei

spp. paracasei 1 SKG 44 ie, 0%, 2%, 4%, 6%, and 8% were used in this study. Each treatment was repeated

three times to get 15 experimental units. The data obtained were analyzed by ANOVA, and the significant

differences effect were followed by Duncan Multiple Range Test (DMRT). The best effect resulted by 2%

concentration of Lactobacillus paracasei spp. paracasei 1SKG44, that contain of: 12.26% cheese result, pH

4.31; 54.62% water, 26.57% fat; 24.62% protein; LAB population of 3.9x108 cfu / g, and accompanied by

panelists acceptancy to. : flavor, taste, color, and overall favored.

1 INTRODUCTION

1.1 Background

Cheese is a dairy product made through milk

clotting process using acid or rennet which made

from the abomasum extract (Purnomo in Permainy

et al, 2013). Rennet preparation take a long time and

expensive process, as well as raw material

(abomasum) from cow, therefor needs coagulant

alternatif as good as rennet. Alternative coagulants

that have been studied include pineapple, lime and

starfruit (Sumarmono and Suhartati, 2012), fruit of

the Solanum dubium, sap of Sideroxylon

obtusifolium (Silva et al, 2013) and bark of rampelas

(Silva, 2010; Suardika, 2015).

The bark of Rampelas (BR) has long been used

by the East Timor leste community as a clotting milk

to produce a traditional food known as Ami Maka-

Ana (AMA) (Silva, 2010). The tannin compound in

BR is thought to be able for clotting cacein, so that it

can be used as a natural coagulant instead of rennet

(Santini, 2014). The use of extract BR as a cheese

coagulant has several disadvantages, such as a small

yield (9.8%) and a distinctive cheese flavor

(Suardika, 2015). Therefore it is necessary the

addition of lactic acid bacteria to correct these

deficiencies.

Lactobacillus paracasei spp. paracasei 1 SKG44

is an isolated lactic acid bacteria from wild horse

milk, was chosen because it strain of indegenous

LAB, thought potentially to be used as a probiotic

because it has the ability to inhibit pathogenic

bacteria and has been tested as a curd starter, both in

the form of dry (powder) and wet (Sugitha, 2008).

Therefore, in this study, the application of

Lactobacillus paracasei spp paracasei 1 SKG44 to

quality improvement of cheese with extract

coagulant was made lightly. This study aims to

determine the additional effect of Lactobacillus

paracasei spp. paracasei 1 SKG44 to improve the

quality of cheese.

2 MATERIALS AND METHODS

2.1 Place and Time of Research

This research was conducted at Integrated

Laboratory of Bioscience and Biotechnology

(ILBB), Udayana University; Food Analysis

Laboratory; Biochemistry and Nutrition laboratory

of Food Science and Technology Department,

210

Sugitha, I., Damayanti, T. and Artha, K.

Application of Lactobacillus paracasei spp. Paracasei 1 SKG 44 to Improve Cheese Quality with Extract of Rampelas (Ficus ampelas Burm F).

DOI: 10.5220/0010001200002964

In Proceedings of the 16th ASEAN Food Conference (16th AFC 2019) - Outlook and Opportunities of Food Technology and Culinary for Tourism Industry, pages 210-215

ISBN: 978-989-758-467-1

Faculty of Agricultural Technology, Udayana

University

2.2 Tools and Materials

The tools used in this study were test tubes (Iwaki

pyrex), petri dishes (Duran), measuring cups (Iwaki

pyrex), beaker cups (Iwaki pyrex), erlenmeyer

(Iwaki pyrex), centrifuse tube (Falcon),

microcentrifuge tube (Nesco ), micropippete

(Gilson), incubator (Memmert BE 400), autoclave

(Tomy ES 315), Laminar Flow Cabinet (ESL JSCB-

900SB), Centrifuse (Clement GS 150), Waterbath

(Eyela SB 35), blue tips (QSP), yellow tips (QSP),

The material to be used in this study is milk

(Greenfield Fresh Milk) which was purchased at a

local supermarket, isolate Lactobacillus paracasei

spp. paracasei 1SKG44, which has been stored in

30% glycerol at ILBB. The chemicals used The

Mann Rogosa Sharpe Agar (MRSA) (Pronadisa), de

Mann Rogosa Sharpe Broth (MRSB) (Pronadisa),

0.85% NaCl (Merck), 50% NaOH (Brataco), H2SO4

(JT Baker), 1N HCl (JT Baker), Aquades (Chemical

Sabha), Petroleum Ether (Bratachem), 3% Boric

Acid (Merck), Kjeldahl Tablets, Phenolphthalein

Indicators, Kitchen Salt (Rafina), and CaCl2 (Merck

2.3 Research Design

The completely randomized design (CRD) wa used

in the research with a concentration of 25% bark

extract extract (Suardika, 2015) and the

concentration of Lactobacillus paracasei spp.

paracasei 1SKG44 which consists of 5 levels (v / v),

such as :1=0%;2= 2%;3= 4%;4= 6% and 5= 8%.

Each treatment was repeated 3 times to obtain

15 experimental units. The data obtained were

analyzed by ANOVA and if there were significant

effects should continue by the Duncan Multiple

Range Test (DMRT) test, The variables observed in

this study were recovery, pH, water content, total

lactic acid bacteria, protein content, fat content, and

sensory properties

2.4 Research Implementation

Stock isolates Lactobacillus paracasei spp.

paracasei 1SKG44 was inoculated as much as 50 μl

into 10 ml MRSB media and incubated at 37 ° C for

24 hours. Then confirmation to gas and catalase test,

Gram staining (Dewi et al, 2014) and morphological

observation. The mass production of cells by

transferring 10 ml of MRSB which was positively

overgrown with isolates into 100 ml of sterile

MRSB, and incubated at 37 ° C for 24 hours, then

inserted into several centrifuge tubes, at 3500 rpm

for 10 minutes separating pellets and supernatants.

Once separated, the supernatant was removed and

the pellets washed using saline solution 0.85% 1

times, and re-centrified. Pre-washed pellets are

inoculated into milk.

The making of crude bark extract was done by

extraction method (Santini, 2014). The 60 mesh

sifted sample was weighed 160 g and inocluded in to

1000 ml erlemeyer. The result of sandpaper

(powder) was dissolved with a 960 ml water solvent

which was heated at 90 ° C. Comparative

comparison with solvent is 1: 6. A sanding powder

solution is heated to a temperature of 90 ° C, and left

for 15 minutes. The solution is filtered by using

Whatman filter paper No.1. Extract obtained is

placed in a dark bottle.

Cheese making is done according to the modified

method of Law and Tamime (2010). 400 ml of milk

for each treatment was heated to 80 ° C, then the

bark of 100% (25%) was taken into account and the

temperature was maintained for 30 minutes and then

cooled to temperature (± 30 ° C) and inoculated

Lactobacillus paracasei spp. paracasei 1SKG44

according to treatment (population 10

CFU / ml).

Milk was then incubated for 18 hours at 41 ° C.

After producing curd the whey was separated, curd

is cut into small pieces, then scalding or cooking at

40 ° C for ± 1 hour while stirring. Then separation of

curd and whey was done using cheese cloth, and

whey was left to drip for several minutes. Strain

strips containing curd are then inserted into the mold

and pressed using a load of ± 300g for 24 hours.

Printed cheese, then soaked (Brining) with a solution

consisting of sterile water, 3% kitchen salt and

CaCl

0.3% for 24 hours, then drained and analyzed

3 RESULTS AND DISCUSSION

3.1 Cheese Quality

Nutritional composition and total LAB of Cheese

produced of coagulant extract and the treatment of

Lactobacillus paracasei spp. paracasei 1SKG 44 can

be seen in Table 1.

Application of Lactobacillus paracasei spp. Paracasei 1 SKG 44 to Improve Cheese Quality with Extract of Rampelas (Ficus ampelas Burm

211

Table 1: Average nutritional composition and total LAB cheese of the sample.

Treatment Yield(%) pH Water(%) Fat(%) Protein(%) Total LAB (cfu/g)

KB1 12,87 a 5,35 d 55,45 a 26,67 b 25,27 a 5,7x10

± 0,27

KB2 12,26 a 4,31 c 54,62 a 26,57 b 24,65 a 3,9x10

± 0,89

KB3 13,12 a 4,03 b 52,72 a 22,06 ab 23,6 a 5,2x10

± 0,82

KB4 14,44 a 3,81 a 54,19 a 23,68 ab 24,32 a 1,07x10

± 0,59

KB5 16,9 b 3,78 a 62,27 b 19,7 a 26,27 a 2,8x10

± 0,62

Description: KB1 = 0%, KB2 = 2%, KB3 = 4%, KB4 = 6%, KB5 = 8%. The same letter behind the average value in

the same column shows a non-significant effect (P> 0.05)

3.1.1 Recovery

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 had a significant effect (P

<0.01) on the yield of cheese produced. Table 1

shows the highest yield average of cheese obtained

in KB5 treatment with a concentration of 8%. The

cheese yield has a tendency to increase with

increasing isolate concentration, although

statistically the KB1 to KB 4 treatment has no

significant effect (P> 0.05). This shows that even

though tannins in extracts and Lactobacillus

paracasei spp. paracasei 1 SKG44 has a different

mechanism of milk clotting, but the use of both can

increase the yield of cheese at a concentration of

25% extract and 4-8% Lactobacillus paracasei spp.

paracasei 1 SKG44. Addition of lactic acid bacteria

such as Lactobacillus paracasei spp. paracasei 1

SKG44 causes a decrease in pH, increases the

degree of syneresis, and the release of calcium

phosphate colloid from casein (Law and Tamime,

2010). Polished babakan extract contains tannin

compounds that can agglomerate milk protein to

produce cheese (Suardika, 2015). According to

Makkar et al (2007) in Firdausi et al (2013) tannin

compounds contained in plants naturally have the

ability to interact with proteins and form complex

proteins

3.1.2 pH

Based on Table 1, the pH of cheese tends to

decrease, along with the increasing concentration of

LAB used. The decrease in pH was statistically high

significant in each treatment (P <0.01), only in the

KB4 and KB5 treatments the difference was not

significant (P> 0.05). This shows that the higher the

concentration of bacteria, the lower the pH produced

due to the increasing activity of LAB in the process

of lactose fermentation to lactic acid. According to

Walstra et al (2006), the percentage of inoculums

which are higher will increase acid production and a

decrease in pH value can increase the degree of

syneresis. Lactic acid is responsible for acid flavors

in raw cheese and plays an important role in the

formation and texture of curd, synthesis of

proteolytic and lipolytic enzymes involved in

ripening cheese, and suppressing pathogenic

microbes and spoilage microorganisms (Jamilatun,

2009)

3.1.3 Water Content

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 had a significant effect (P

<0.05) on the average water content of cheese

produced. The results showed that the increase in

water content in each treatment tended to change. In

the KB1, KB2, KB3 treatment the water content

tends to decrease. In the KB4 and KB5 treatment an

increase in water content. Statistically, KB1 to KB4

does not have a real effect. The KB5 treatment has

the highest effect and the highest water content,

which is 62.27%. Cheese made by using acid as a

clot, such as lactic acid as a result of lactic acid

bacteria fermentation, is classified into acid-

coagulated cheese. Lactic acid bacteria such as

Lactobacillus paracasei spp. paracasei 1 SKG44

ferments lactose into lactic acid, acidifies milk and

collects casein micelles into a net matrix, and

converts Micellar Calcium Phosphate (MCP) to a

soluble form (Kinstedt, 2014). The more

concentration of Lactobacillus paracasei spp. added

paracasei 1 SKG44, water content tends to increase.

This is consistent with the statement of Lucey

(2004) in Kinstedt (2014) that casein matrices which

lose MCP tend to be more difficult to release whey,

therefore, acid-coagulatedcheese has a higher water

content.

3.1.4 Fat Level

The analysis showed that the concentration of

Lactobacillus paracasei spp. paracasei 1 SKG44

significantly affected (P <0.05) on the average fat

content of the cheese produced. The average fat

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content tends to decrease as more bacterial

concentrations are inoculated. Giving 0% and 2%

Lactobacillus paracasei spp. paracasei 1 SKG44

has no significant effect on fat content. Decrease in

fat levels began to be seen at the 4% and 6%

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 and significantly decreased at

athe concentration of 8%. Bottazi (1983) in

Sunarlim et al (2007) states that lactic acid bacteria

have lipolytic activity, which can break down milk

fat into simple chemical compounds. The higher the

concentration of bacteria, the lower the fat content of

cheese because the increases activity of lactic acid

bacteria.

3.1.5 Protein Levels

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 had no significant effect (P>

0.05) on the average levels of cheese protein

produced. Addition of Lactobacillus paracasei spp.

paracasei 1 SKG44 at a concentration of 0%, 2%,

4%, 6%, and 8% gave a non-significant effect on the

levels of cheese protein produced. Based on research

by Mardiani et al. (2013), increasing the

concentration of LAB which will increase the level

of protein in cheese, because LAB has a protease

enzyme that breaks down protein into amino acids.

In cheese making with the coagulant extract, the

tannin is extracted, the tannin has the ability to bind

to proteins forming protein-tannin compounds.

Tanin naturally has the ability to inhibit the action of

enzymes (enzyme inhibitors)(Smith et al., 2005;

This was thought to cause Lactobacillus paracasei

spp. paracasei 1 SKG44 cannot break down cheese

protein so that the levels of protein produced are not

significantly affected

3.1.6 Total Lactic Acid Bacteria

The total calculation of lactic acid bacteria shows

that the total LAB per 1 gram of cheese increases

with the concentration of Lactobacillus paracasei

spp. paracasei 1 SKG 44 added. The KB1 treatment

which did not get the addition of Lactobacillus

paracasei spp. paracasei 1 SKG44 produced an total

average LAB of 5.7 x 10

. This number is greater

than the total average LAB of Ami-Maka Ana

(Sugitha et al 2014), which ranges from 0.88 x 10

to 5.36 x 10

. This is thought to be caused by natural

lactic acid bacteria in milk which still survive during

the pasteurization and develop during the

fermentation process. The concentration of bacteria

added to KB2, KB3, KB4, and KB5 treatments

sequentially are 2%, 4%, 6%, 8%, with the

population of Lactobacillus paracasei spp.

paracasei 1 SKG44 ± 1 x 10

cfu / ml. The total

lactic acid bacteria shown in Table 1 represents the

viabilitias of Lactobacillus paracasei 1 spp.

paracasei SKG44 during the cheese making process.

3.2 Sensory Analysis

The results of the analysis datas showed in Table 2.

3.2.1 Color

The analysis showed that the concentration of

Lactobacillus paracasei spp. paracasei 1 SKG44 had

a very significant effect on the color of the cheese

produced (P <0.01). In the KB1 treatment showed a

significant difference (P <0.05) compared to other

treatments with the color of yellow cheese, while the

KB2, KB3, KB4, and KB5 treatments were not

significantly different (score 4 = yellowish white).

The color of cheese is influenced by the color of

milk do to carotene pigments that dissolve in fat

(Jamilatun, 2009

3.2.2 Flavour

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 has a very significant effect on

the flavour of cheese produced (P <0.01). The KB1

treatment showed significant differences (P <0.05)

with a scent test scale of 2.46 (not typical of cheese

Flavour). The KB2 treatment produces the highest

Table 2: The score average of sensory cheese analysis of the sample.

Treatment Color Flavor Texture Taste Acceptace

KB1 3.87 a 2.46 a 4.2a 2.06 a 2.33 a

KB2 4.2 b 4.06 b 4.13a 3.33 b 3.53 b

KB3 4.33 b 3.46 b 4.00a 3.20 b 3.46 b

KB4 4.33 b 3.53 b 4.20a 3.40.b 3.20 b

KB5 4.47 b 3.46 b 4.40a 3.13 b 3.46 b

Note: KB1=0%, KB2=2%, KB3=4%, KB4=6%, KB5=8%. Superscript in the same colom was not

significant different (P>0.05)

Application of Lactobacillus paracasei spp. Paracasei 1 SKG 44 to Improve Cheese Quality with Extract of Rampelas (Ficus ampelas Burm

213

Flavour). The KB2 treatment produces the highest

Flavour sensory value, which is 4.06 or somewhat

typical of the Flavour of cheese. The treatment of

KB3, KB4, and KB 5 was not significantly different

(P> 0.05) with a scent test scale 3 (usual). In

generally, the taste and Flavour of cheese appeared

due to the volatile component formed after starter

microbial inoculation which could result in

biochemical changes include proteolysis, lipolysis,

and lactose fermentation.

3.2.3 Texture

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG 44 did not significantly affect the

texture of the cheese produced (P> 0.05). In this

study, panelists were asked to assess the texture of

cheese by pressing cheese with the fingertips. The

results showed that KB1, KB2, KB3, KB4, and KB5

were not significantly different from the 4 (rather

soft) texture test scale. The formation of texture is

influenced by lactic acid produced by lactic acid

bacteria. Lactic acid is a metabolite of lactic acid

bacteria. Lactic acid can agglomerate milk protein

through a mechanism for decreasing pH. Texture

can be interpreted as an attribute of cheese resulting

from a combination of physical attributes, including

the size, shape, number and adjustment of structural

elements perceived by a combination of the sense of

taste, sense of sight and sense of hearing.

3.2.4 Taste

The results of the analysis showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG 44 had a very significant effect on

the taste of cheese produced (P <0.01). In the taste

test hedonic test (preference) was carried out. The

KB1 treatment was significantly different (P> 0.05)

with a taste test scale 2 (disliked). Whereas KB2,

KB3, KB4, and KB5 treatments were not

significantly different (P <0.05) with taste test scale

3 (usual). Flavor is built by starter culture, and

enzymatic modification of various components of

milk. Lipolysis is very important in the formation of

the flavor of the cheese. Bacterial starters have a

direct effect on fresh cheese taste, for example

cottage cheese. In the process of making cheese

there is also a process of adding salt consisting of

3% salt and CaCl2 0.3%. Salt was added in the form

of crystals which have been dissolved in sterile

water.

3.2.5 Overall Acceptance

The ANOVA test results showed that the

concentration of Lactobacillus paracasei spp.

paracasei 1 SKG44 had a very significant effect on

the overall acceptance of cheese produced (P <0.01).

The KB1 treatment received the smallest evaluation

from the panelists, namely 2.33 (somewhat dislike).

The treatment of KB2, KB3, KB4, and KB5 is

assessed on a scale of 3 (normal). This shows that

cheese with the addition of Lactobacillus paracasei

spp. paracasei 1 SKG44 can be accepted by

panelists.

4 CONCLUSION

Based on the research that has been done, it can be

concluded as follows:

 Addition of Lactobacillus paracasei spp.

paracasei 1SKG44 in making cheese with bark

extract of rampelas can improve cheese quality

and highly significant effect on yield, pH,

sensory color, Flavour, taste and overall

acceptance, and significantly affect the water

content and fat of cheese, but not significantly

affect to protein.

 The Treatment of the addition of Lactobacillus

paracasei spp. paracasei 1 SKG44 is KB2 with

a yield of 12.26%; moisture 54.62%; fat

26.57%; pH 4.03; protein 24.65% and total

LAB 3.9x10

cfu / g with the highest sensory

value among other treatments.

 Suggestion for further research is needed

regarding the shelf life, probiotic of cheese with

coagulants of rampelas bark extract and

Lactobacillus paracasei spp. paracasei

1SKG44.

ACKNOWLEDGEMENTS

This paper is part of a study entitled: Optimization

of the Manufacture of Traditional Soft Cheese (Soft

Cheese) by using Rampelas (Ficus Ampelas) and

Lactic Acid Bacteria as Natural Coagulants; funded

through the Directorate of Research and Community

Service, Directorate General of Higher Education,

Ministry of Education and Culture. Thank you for

your help, and hope this article is useful.

16th AFC 2019 - ASEAN Food Conference

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