Quantification of Lactic Acid as Secondary Metabolite of Lactic Acid

Bacteria Isolated from Milk and Its Derivatived Products

Fifi Afiati

, Fitri Setiyoningrum

, Gunawan Priadi

and Vania Qyasaty

RC Biotechnology – LIPI, Jl. Raya Bogor, Km. 46 Cibinong 16911, Indonesia

Brawijaya University, Malang, Indonesia

Keywords: Lactic Acid, Lactic Acid Bacteria, Total Acid Test, Secondary Metabolite.

Abstract: Lactic acid is a widely used secondary metabolite product. Titrated Total Acid (TTA) analysis is one of

method to learn quantification process of lactic acid as a secondary metabolite of Lactic Acid Bacteria (LAB).

The TTA test was carried out on 158 isolates from 11 samples with 2 replications using culture supernatant.

The test was started with preparing growth media then rejuvenating the culture. NaOH 0.1 N, oxalic acid 0.1

N, and PP 1% indicator were prepared as reagents for analysis. As the result, the smallest TTA value is shown

in the 1-Sa-L sample with a TTA value of 0.41% and the largest TTA value is shown in the KGL-5 sample

with a TTA value of 1.56%.

1 INTRODUCTION

Lactic acid bacteria (LAB) is common used bacteria

as starter to produce food product such as yoghurt,

cheese, and sausage. LAB has food equivalent safety

(food grade). LAB could reproduce in digestive tract,

affect balance of digestive tract bacteria which

provide a healthy effect, as probiotic. LAB is isolated

from various sources and explored functional

potential effect to enhance human health. Milk is rich

of protein, fat, carbohydrate (mainly lactose), and

vitamin and mineral (Park et al., 2007), and become

ideal habiTTA for microorganism growth. Lactose, as

main carbohydrate compound in milk, is fermented

with microorganism, especially LAB group to

produce lactic acid as major metabolite, creating milk

with sour taste. Lactic acid is secondary metabolite

product, could be produced in two ways which are

using chemical synthesis and microbiological

fermenTTAion.

Lactic acid production using microbiological

fermenTTAion has some advantages, one of the

advantage is the high purity (90 – 95 %)

(Kotzamanidis et al., 2002) of optical L(+) lactic acid

with high crystallinity and melting point. In the other

hand, chemical synthesis of lactic acid produce mix

result in D-L configuration. FermenTTAion in

producing lactic acid has some disadvantages such as

bacteria growth medium is not economical since it

contains some expensive composition like yeast

extract and peptone. FermenTTAion process

produces primary and secondary metabolite. Primary

metabolite is chemical compound produced and used

by microbes to regenerate which are lactic acid and

alcohol. Secondary metabolite is compound produced

by microbes but not used for physiological activity

like bacteriocin. Kefir fermenTTAion is done with

LAB and yeast. LAB is used to produce lactic acid

from glucose and triggered yeast’s growth. In kefir

making process, yeast has function to produce ethanol

and flavor component as specific kefir taste. (Usmiati,

2007)

Whey contains high nutrition and has benefit for

health such as controlling body metabolism,

probiotic, animal antitumor, and antibacterial

(Farnworth, 2003). Whey is defined as serum or the

water portion of the milk that remains after separation

of curd (cheese) and is the result of coagulation of

milk protein with acids and proteolytic enzymes

(Usmiati, 2007). One kilogram of cheese produced

will produce as much as 8 to 10 liters of whey

(Farnworth, 2003). Whey in Indonesia is generally

underutilized, thus polluting the environment. Whey

cheese has a Biochemical Oxygen Demand (BOD)

and Chemical Oxygen Demand (COD) content which

does not meet the permitted safe limits, namely

50,000 mg / L (BOD) and a COD level of 80,000 mg

/ L (Guimaraes et al., 2010). Environmental pollution

can be reduced by utilizing whey as a basic ingredient

132

Aﬁati, F., Setiyoningrum, F., Priadi, G. and Qyasaty, V.

Quantiﬁcation of Lactic Acid as Secondary Metabolite of Lactic Acid Bacteria Isolated from Milk and Its Derivatived Products.

DOI: 10.5220/0010546700003108

In Proceedings of the 6th Food Ingredient Asia Conference (6th FiAC 2020) - Food Science, Nutrition and Health, pages 132-136

ISBN: 978-989-758-540-1

in the manufacture of probiotic functional drinks such

as yogurt or kefir. Whey kefir is the latest innovation

in whey processing which is fermented by a number

of microbes, namely lactic acid-producing bacteria

(LAB), acetic acid-producing bacteria, and yeast.

Inhibition zone produced by the metabolite

compound L. plantarum against Gram-negative and

positive pathogenic bacteria is a result of the presence

of primary metabolite such as lactic acid, ethanol,

diacetyl, and carbon dioxide and secondary

metabolites such as bacteriocins and hydrogen

peroxide.

Inhibition zone produced by the metabolite

compound L. plantarum against Gram-negative and

positive pathogenic bacteria is a result of the presence

of primary metabolite such as lactic acid, ethanol,

diacetyl, and carbon dioxide and secondary

metabolites such as bacteriocins and hydrogen

peroxide.

Logarithmic phase is 15-24 hours on incubation

period. Metabolite compounds from L. plantarum can

inhibit Gram-positive and negative pathogenic

bacteria which are often found in foodstuffs of animal

origin, especially meat and milk, so that it has a good

enough potential to be studied in more depth

regarding its use directly in foodstuffs of animal

origin. The process of producing bio preservation

materials for food from the metabolism of L.

plantarum has the potential to be developed and

applied to foodstuffs of animal origin, especially milk

and meat.

2 MATERIALS AND METHODS

2.1 Preparation of Growth Media and

LAB Culture Growth

The growth media for Lactic Acid Bacteria (LAB) are

MRS-agar and MRS-broth. Sterile Lactobacillus

MRS Broth (HIMEDIA, M369) was used. Pre-culture

stage was needed to refresh isolates which used to be

stored at -80 °C before use, then isolates were

incubated for 24 hours at 37 °C, characterized by a

white sediment and the growing medium becomes

cloudier. After that, isolates were homogenized and 5

µL was regrown in sterile MRS Broth media to be

incubated for 18 hours (overnight) at 37 °C

2.2 Total Titrated Acid Test (TTA)

One of the reagents in the Titrated Total Acid (TTA)

test is 0.1 N NaOH. Volume of NaOH needed

showing the acid present in isolates because NaOH

would neutralize the acid and was indicated by a

change in color to pink.

The amount of NaOH and distilled water used in

order to get the desired normality follows the

following formula:

NaOH =

ram/MR × 1000/(v (mL)) (1)

2.3 Preparation of 0.1 N Oxalic Acid

Preparation of 0.1 N Oxalic Acid solution aims to

standardize and determine the normality of NaOH

precisely. Weighed 0.9 gram of powdered oxalic acid

and dissolved in 100 mL of distilled water then stirred

until the solids and solution were completely mixed

to form a 0.1 N oxalic acid solution. The calculation

of the amount of powdered oxalic acid with a relative

molecular mass value of 90 (Mr = 90) and the distilled

water used in order to obtain the desired normality

follows the following formula:

oxalic acid =

ram/MR × 1000/(v (mL)) (2)

2.4 Standardization of NaOH

NaOH standardization is carried out to determine the

normality value of NaOH in more detail. NaOH

standardization is carried out by using the titration

principle. The 0.1 N oxalic acid solution was put into

a 100 mL erlenmeyer, while the NaOH solution was

put into a burette. The 0.1 N oxalic acid solution was

then titrated with NaOH solution until it turned pink.

Titration is carried out three times so that the results

can be more accurate. At each titration, the volume of

NaOH used was recorded. The volume of NaOH

obtained is then averaged so that the final volume will

be used in the calculation. The standardized value of

NaOH concentration is 0.1135 N. Calculation of the

NaOH concentration follows the following formula:

N NaOH = (Volume oxalic acid × N oxalic

acid)/(Volume NaOH)

(3)

2.5 Preparation of PP (Phenolphtalein)

Indicator 1%

The PP indicator is used as a color indicator in the

titration process both in the total acid test and when

standardizing NaOH. The PP indicator serves to

determine the exact equivalence point in a solution

marked by a change in color. 0.5 grams PP in powder

form is weighed using a digital scale, then dissolved

with 96% ethanol 50 mL in a beaker glass until well

blended. The finished PP indicator solution is then put

Quantiﬁcation of Lactic Acid as Secondary Metabolite of Lactic Acid Bacteria Isolated from Milk and Its Derivatived Products

133

in a dark glass bottle to keep the concentration

unchanged.

TTA test was carried out on 80 samples with twice

analysis each sample. Repetition is purposed to obtain

more accurate results. If the standard deviation

between repetitions does not exceed 10%, the results

obtained can be used as final data. The TTA test was

carried out to determine the acid content found in

LAB isolates. The measurement of the total titrated

acid (TTA) is the determination of the total acid

concentration. Total Titrated Acid (TTA) relates to

the measurement of the total acid contained. The TTA

value includes the measurement of total dissociated

and undissociated acid. The results of TTA

measurement are used to determine the amount of

organic acids in fruits and vegetables.

TTA test is carried out only on the supernatant

part of the culture so that before the TTA test is

carried out, the sample is centrifuged first. The

sample to be used is put into a microtube for

centrifuge. The centrifuge was carried out at a speed

of 9000 rpm for 2 minutes.

TTA test basically uses the titration principle. 1

ml sample is pipetted into a 100 mL erlenmeyer, then

diluted up to 10 times by adding 9 mL of sterile

distilled water. To show the equivalence point in the

solution, the PP indicator is added so that the solution

will turn pink when it reaches the equivalence point.

When it reaches the equivalence point, the titration is

stopped and the initial volume and final volume of

NaOH are recorded which will be used in the

calculation to obtain the final data.

3 RESULTS

The following are the results of the TTA analysis for

each sample:

Table 1: Total Titrated Acid (TTA) value from milk and

its derivated products.

Isolates TTA (%)

K. Kam 1 0.996 ± 0.04

K. Kam 2 1.468 ± 0.09

K. Kam 3 1.124 ± 0.04

K. Kam 4 0.562 ± 0

K. Kam 5 1.660 ± 0.07

K. Kam 6 0.562 ± 0.07

Table 2: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

W.K. Sapi 1 1.532 ± 0

W.K Sapi 2 1.545 ± 0.02

W.K Sapi 3 1.162 ± 0.05

W.K Sapi 4 1.328 ± 0.04

W.K Sapi 5 1.187 ± 0.02

W.K Sapi 6 1.315 ± 0.09

Table 3: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

KGG - 1 0.664 ± 0

KGG - 2 0.6 ± 0.02

KGG - 3 0.626 ± 0.05

KGG - 4 0.613 ± 0

ABC

KGG - 5 0.562 ± 0

KGG - 6 1.622 ± 0.02

KGG - 7 1.609 ± 0.04

KGG - 8 0.626 ± 0.02

Table 4: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

KGP - 1 0.562 ± 0.04

KGP - 2 0.6 ± 0.05

KGP - 3 1.2 ± 0.07

KGP - 4 0.626 ± 0.02

KGP - 5 1.302 ± 0.07

KGP - 6 1.111 ± 0.09

KGP - 7 1.124 ± 0

KGP - 8 1.175 ± 0.07

KGP - 9 0.702 ± 0.09

Table 5: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

KGB - 1 0.702 ± 0.02A

KGB - 2 0.753 ± 0.02AB

KGB - 3 0.728 ± 0.02AB

KGB - 4 0.766 ± 0.04AB

KGB - 5 1.251 ± 0.07F

KGB - 6 1.149 ± 0EF

KGB - 7 0.958 ± 0.09CD

KGB - 8 1.047 ± 0DE

KGB - 9 0.856 ± 0.09BC

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134

Table 6: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

KGL - 1 1.468 ± 0.02A

KGL - 2 1.073 ± 0B

KGL - 3 1.085 ± 0.05B

KGL - 4 0.741 ± 0.04A

KGL - 5 1.558 ± 0.07A

KGL- 6 1.047 ± 0.04B

KGL- 7 0.729 ± 0.04A

KGL- 8 1.366 ± 0.02C

KGL- 9 1.302 ± 0.07C

Table 7: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

SS - 1 1.353 ± 0.04E

SS - 2 1.047 ± 0D

SS - 3 0.843 ± 0.04C

SS - 4 0.536 ± 0A

SS - 5 0.779 ± 0.02B

Table 8: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

1-Sa-A 0.511 ± 0.04A

1-Sa-B 0.677 ± 0.09B

1-Sa-E 0.485 ± 0.04A

1-Sa-F 0.447 ± 0.02A

1-Sa-G 0.421 ± 0.02A

1-Sa-H 0.498 ± 0.09A

1-Sa-I 0.421 ± 0.05A

1-Sa-J 0.498 ± 0.02A

1-Sa-

0.409 ± 0.04A

1-Sa-L 0.409 ± 0.04A

Table 9: Total Titrated Acid (TTA) value from milk and its

derivated products.

Isolates TTA (%)

2-Sa-A 0.562 ± 0.07A

2-Sa-B 0.715 ± 0B

2-Sa-C 1.073 ± 0.07C

2-Sa-D 0.434 ± 0.04A

Table 10: Total Titrated Acid (TTA) value from milk and

its derivated products.

Isolates TTA (%)

2-JR-A 0.485 ± 0A

2-JR-B 0.498 ± 0.02A

2-JR-C 0.524 ± 0.02AB

2-JR-D 0.511 ± 0.04AB

2-JR-E 0.562 ± 0.04BC

2-JR-F 0.536 ± 0.04ABC

2-JR-G 0.587 ± 0C

Table 11: Total Titrated Acid (TTA) value from milk and

its derivated products.

Isolates TTA (%)

1-PE-A 1 0.69 ± 0.04C

1-PE-C2 0.664 ± 0BC

1-PE-D3 0.447 ± 0.05A

1-PE-E4 0.562 ± 0.07AB

1-PE-G5 0.549 ± 0.02AB

1-PE-I 6 0.932 ± 0.05D

According to Kusumawati et.al (2008), lactic acid

bacteria produce compounds that are antimicrobial

such as organic acids, hydrogen peroxide and protein

compounds (bacteriocin). Lactobacilli species which

produce sufficiently large amounts of hydrogen

peroxide can inhibit growth and kill pathogenic

microbes. The accumulation of these compounds in

cells occurs because lactobacilli does not produce the

enzyme catalase.

Lactic acid bacteria will convert carbohydrates

into lactic acid under anaerobic conditions and this

process can be divided into three stages. In the early

stages, starch from carbohydrate sources will be

hydrolyzed into maltose by α and β amylase, then this

maltose molecule will be broken down into glucose

by maltase and in the final stage lactic acid bacteria

will convert glucose into lactic acid and other

materials such as acetic acid and alcohol.

Secondary metabolites are compounds that are

synthesized by microbes but are not a basic

physiological requirement. One of the secondary

metabolites that can function as antibacterial is

bacteriocin (a protein compound that exhibits

antibacterial activity and is able to prevent the growth

of disease-causing bacteria). Bacteriocin produced in

the decay or stationary phase, which is the phase

when the substrate starts to run out, will stimulate the

formation of enzymes that play a role in the formation

of secondary metabolites.

Lactic acid bacteria (LAB) are a group of bacteria

that have been widely used as a starter to produce

food ingredients such as yogurt, cheese and sausages.

Based on a long history of safety in the consumption

of food produced using LAB, this class of bacteria is

recognized as having food-grade safety. LAB isolates

were characterized morphologically and

physiologically based on Gram characteristics, gas

production from glucose and catalase production.

Only Gram positive and catalase negative isolates

were further identified. Catalase testing is done by

dropping one drop of LAB culture over 30% H2O2

solution. Positive catalase is characterized by the

formation of foam (bubbles / foam). Gram staining

was carried out to see cell morphology and

Quantiﬁcation of Lactic Acid as Secondary Metabolite of Lactic Acid Bacteria Isolated from Milk and Its Derivatived Products

135

characteristics of Gram. Gas formation from glucose

is carried out using hot loops and gas formation is

characterized by the formation of froth. Lactic acid

bacteria also produce other metabolites that function

as anti-microbes such as acetic acid, hydrogen

peroxide, and bacteriocins. The increase in lactic acid

from secondary metabolites of lactic acid bacteria is

caused by the increasing number of cell biomass that

ferments the substrate into lactic acid and energy.

According to Yusmarini and Efendi (2004), the sugar

contained in the media is used by lactic acid bacteria

as a carbon source to produce lactic acid and energy

through the glycolysis process.

According to Farnworth (2003), fermentation of

milk into kefir produces metabolites that are

beneficial to health, namely exopolysaccharides and

bioactive peptides. Both of these compounds will

stimulate the immune system. The polysaccharides

formed in kefir also act as anti-tumor agents.

Antibacterial components are also produced during

kefir fermentation such as organic acids (lactic and

acetic acids), carbon dioxide, hydrogen peroxide,

ethanol, diacetyl, and peptides (bacteriocins) which

are not only useful for inhibiting the growth of

pathogenic bacteria and spoilage bacteria during food

processing and storage, but can also be used for the

prevention of some digestive disorders and infections.

The composition and taste of kefir will differ

significantly depending on several factors. The main

factor that can distinguish is the source of milk used

whether it comes from cow's milk or goat's milk.

Other factors that play a role are the fat content of the

milk used, the composition of the kefir seeds and

starter used, and the accompanying technological

process. Traditionally kefir is produced by adding

kefir seeds to some milk.

REFERENCES

Farnworth, E.R. 2003. Handbook of Fermented Functional

Foods. CRC Press. New York

Guimaraes P.M.R., J.A. Teixeira, L. Domingues. 2010.

Fermentation of lactose to bio- ethanol by yeast as part

of intregated solution for the valorisation of cheese

whey. Journal Elsevier. 28(1): 375-384.Hidayatulloh,

A, J Gumilar, E Harlia. 2019. Potensi Senyawa

Metabolit Yang Dihasilkan Lactobacillus Plantarum

Atcc 8014 Sebagai Bahan Biopreservasi Dan Anti

Bakteri Pada Bahan Pangan Asal Hewan. JITP.

7(2):6pp

Kotzamanidis, C. H., Roukas, T., & Skaracis, G. (2002).

Optimization of lactic acid production from beet

molasses by Lactobacillus delbrueckii NCIMB 8130.

World Journal of Microbiology and Biotechnology,

18(5), 441-448.

Park YW, Ju’arez M, Ramos M, Haenlein GFW. 2007.

Physicochemical characteristics of goat and sheep milk.

Small Rumin Res 68: 88-113.

Usmiati, S. 2007. Kefir susu fermentasi dengan rasa

menyegarkan. Warta Penelitian dan Pengembangan

Pascapanen Pertanian. 29(2): 12-13.

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