The Effect of Adding Avocados (Persea americana) on Microbiological and Chemical Qualities in Yoghurt Drinks

Abstract

Yoghurt is one of the fermented products from cow's milk and skim milk as a source of protein to cover nutritional needs. With the development of innovation and the creation of food products, yogurt has many flavors from various fruits and vegetables. Yogurt food product innovations can also be made from skim milk combined with the addition of avocados. Avocado is a fruit that contains fiber, riboflavin (B2), niacin (B3), vitamins A, C, E, K, vitamin B6, magnesium, glutathione, and unsaturated fatty acids. The purpose of this study was to describe the effect of the concentration of avocados (Persea americana) on the quality of microbiology (total LAB) and chemistry (pH, fat content, and protein content) in yogurt drinks. The research used laboratory experimental methods, and data analysis was done descriptively. The treatment of adding avocados is 0%, 10%, 15%, and 20%. The results of the study obtained the effect of the concentration value of adding avocados (Persea americana) with the results of the study: the total value of LAB increased from 4.1x109±0.20 CFU/mL (10%) to 4.4x109±0.07 CFU/mL (15%), the pH value decreased from 4.30±0.02 (0%) to 3.89±0.01 (20%), The fat content value increased from 0.05±0.007 % w/b (0%) to 0.57±0.005 % w/b (20%), the protein content value decreased from 3.46±0.02% w/b (0%) to 2.94b±0.00 % w/b (20%). The conclusion of this study is the effect of adding avocados (Persea americana) on the quality of microbiology (total LAB) and chemistry (pH, fat content, and protein content) in yogurt drinks.

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Jurnal Pijar MIPA
ISSN 1907-1744 (Print)
https://jurnalfkip.unram.ac.id/index.php/JPM
ISSN 2460-1500 (Online)
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How to Cite:
Fatimah, S. A., & Agustini, R. (2024). The Effect of Adding Avocados (Persea americana) on Microbiological and Chemical Qualities in Yoghurt
Drinks . Jurnal Pijar Mipa, 19(3), 493–498. https://doi.org/10.29303/jpm.v19i3.6601
The Effect of Adding Avocados (Persea americana) on Microbiological and Chemical
Qualities in Yoghurt Drinks
Siti Alihatus Fatimah, Rudiana Agustini*
Chemistry Department, State University of Surabaya, Surabaya, Indonesia
*E-mail: rudianaagustini@unesa.ac.id
Received: February 28, 2024. Accepted: April 30, 2024. Published: May 25, 2024
Abstract: Yoghurt is one of the fermented products from cow's milk and skim milk as a source of protein to cover nutritional
needs. With the development of innovation and the creation of food products, yogurt has many flavors from various fruits
and vegetables. Yogurt food product innovations can also be made from skim milk combined with the addition of avocados.
Avocado is a fruit that contains fiber, riboflavin (B2), niacin (B3), vitamins A, C, E, K, vitamin B6, magnesium, glutathione,
and unsaturated fatty acids. The purpose of this study was to describe the effect of the concentration of avocados (Persea
americana) on the quality of microbiology (total LAB) and chemistry (pH, fat content, and protein content) in yogurt drinks.
The research used laboratory experimental methods, and data analysis was done descriptively. The treatment of adding
avocados is 0%, 10%, 15%, and 20%. The results of the study obtained the effect of the concentration value of adding
avocados (Persea americana) with the results of the study: the total value of LAB increased from 4.1x109±0.20 CFU/mL
(10%) to 4.4x109±0.07 CFU/mL (15%), the pH value decreased from 4.30±0.02 (0%) to 3.89±0.01 (20%), The fat content
value increased from 0.05±0.007 % w/b (0%) to 0.57±0.005 % w/b (20%), the protein content value decreased from
3.46±0.02% w/b (0%) to 2.94b±0.00 % w/b (20%). The conclusion of this study is the effect of adding avocados (Persea
americana) on the quality of microbiology (total LAB) and chemistry (pH, fat content, and protein content) in yogurt drinks.
Keywords: Avocado; Fat Content; Protein Content; Total LAB; pH; Yohgurt.
Introduction
Yogurt is a probiotic drink. Yogurt is a processed
fermented milk product made from lactic acid bacteria
(LAB) starter [1]. Yogurt made from skim milk can increase
the nutritional value of yoghurt and provide test results with
better consistency and shape [2]. Avocados (Persea
americana) tend to quickly deteriorate and rot if not handled
carefully after peeling. Combining it with a yoghurt drink is
the best solution to prolong its freshness. Avocado (Persea
americana) is a fruit that contains fiber, riboflavin (B2),
niacin (B3), vitamins A, C, E, and K, vitamin B6,
magnesium, glutathione, and unsaturated fatty acids. One of
the preparations is to make avocado (Persea americana) in
the form of yogurt [3].
The most important factor that makes avocado
(Persea americana) suitable as an additional ingredient in
yogurt is that it can reduce the risk of heart disease. This can
be supported by clinical research published in the Journal of
the American Heart Association; consuming avocado
(Persea americana) (≥ 2x servings per week) is associated
with a reduced risk of cardiovascular disease and the
incidence of coronary heart disease [4]. Based on the Global
Burden of Disease and Institute for Health Metrics and
Evaluation (IHME) 2014 – 2019, heart disease is the highest
cause of death in Indonesia. Basic Health Research
(Riskesdas) data for 2013 and 2018 shows an increasing
trend in heart disease from 0.5% in 2013 to 1.5% in 2018 [5].
Several factors cause an increase in heart disease,
including cholesterol. Cholesterol is also the main cause of
the development of various diseases in the body [6].
Avocado (Persea americana) is a food that contains
unsaturated fat, which can increase high-density lipoprotein
(HDL) [7]. Avocado (Persea americana) contains several
active ingredients that are thought to reduce blood
cholesterol levels, namely niacin (vitamin B3), -sitosterol,
vitamin C, pantothenic acid, oleic acid, Monounsaturated
Fatty Acid (MUFA), folic acid, selenium, amino acids, and
fiber [8]. One type of MUFA often found in nature is omega-
9 (Oleate). Omega-9 fatty acids are found in many nuts,
avocados, and olive oil [9].
Avocado (Persea americana) is suitable as an
addition to yogurt drinks because it contains Omega-9.
Yogurt drinks contain factors that inhibit cholesterol
synthesis, so they can reduce cholesterol levels and prevent
blockage of blood vessels (atherosclerosis), which causes
coronary heart disease [10]. To meet your fat needs,
especially essential fats, you can use skim milk when making
yogurt drinks and add avocado (Persea americana) to it,
which contains oleic-rich oil with a low saturated fat content
and makes avocado oil suitable for direct consumption and
has nutritional support, which is very good and overcomes
cardiovascular disease [11].
This study aimed to describe the effect of increasing
the concentration of avocado (Persea americana) on
microbiological quality (total LAB) using total plate count
(TPC) and chemistry (pH using a digital pH meter, fat
content using Roese-Gottlieb and protein content using
Kjeldahl) yogurt drink. Based on this description, this
research was carried out by making yogurt with adding
avocado (Persea americana) with varying concentrations of
0%, 10%; 15%; and 20%. According to [12], the results of

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May 2024, Volume 19 No. 3: 493-498
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his research showed that the combination of adding soursop
fruit with treatment concentrations of 10%, 15%, and 20%
had the best effect on yogurt, the best impact on adding
soursop fruit juice at a concentration of 10% and a
fermentation time of 12 hours.
Research Methods
Tools and Materials
The tools to be used are glassware, pH meter
(Krisbow-10206579), Infrared Moisture Determination
Balance (FD-610), laminar airflow (Thermo Scientific 1300
Series A2), analytical balance (Denver S1-234),
micropipette (Eppendorf Research Plus), incubator
(Temperature Controller XH-W3001), autoclave (Hirayama
HVE-50), stirrer (DLAB MS7-H550-Pro), centrifugator
(Hrttich EBA 20), vortex (Tab-Net S0200), vortex mixer
(Faithful Xh-D), electric stove (Maspion), fume hood,
pumpkin kjeldahl, measuring flask, condenser, three-neck
flask, stative, clamps, biuret, stopcock, drip pipette,
erlenmeyer, spirtus burner, lighter, spray bottle, knife,
cutting board, blender, strainer, tube rack, petri dish dish,
centrifuge tube, spatula, sterile glass bottle, watch glass,
stirrer magnet.
The materials needed are skim milk (Greenfield),
avocado fruit (Persia americana), Sugar, Starter (Biokul
plain), water, MRSA (Merck), NaCl for analysis (Merck),
Alcohol 70%, CaCO3, pH indicator.
Research Procedure
Avocado Fruit Intake
Taking avocados 3 avocados then cleaned with
running water. Next, separate the avocado from the skin and
seeds, weigh it, and then mash it. Avocado mashed with a
ratio of avocado to water 1:2 using a blender ±3 minutes.
After blending, it is then filtered and placed into a container.
Yoghurt Making
Making yogurt begins with skim milk and is
measured at as much as 400 ml. Skimmed milk is heated at
a pasteurization temperature of ±80ºC. After that, turn off the
stove and wait for the nails to warm. Pasteurized milk is
added with 15% Biokul Plain starter, 8% sugar, and 0%,
10%, 15%, and 20% avocado variations. Then, put it into a
sealed bottle that has been labeled. After that, the
fermentation process is started and put into the incubator at
37°C for 12 hours.
Total Lactic Acid Bacteria (LAB)
Total lactic acid bacteria (LAB) were analyzed using
the Total Plate Count (TPC) method—preparation of 100 ml
NaCl solution, as much as 0.85 grams of NaCl. Then, the
manufacturer of 200 ml MRS A media, as much as 13.4
grams of MRS A, added 1% CaCO3 as much as 1 gram. A
petridish dish is prepared as much as needed, then sprayed
with 96% alcohol. All preparations are put in plastic and an
autoclave for ±3 hours until the temperature drops to 50ºC.
Then, it was incubated in the reverse position for 48 hours at
37ºC with the position of the petridish dish turned upside
down. Growing colonies can be calculated in units of CFU
(Colony Forming Unit)/mL.
Acidity (pH)
Acidity (pH) is analyzed using the digital pH meter
method. The pH meter is calibrated first with pH 4 and pH 7
buffers according to the pH range of yogurt. The
measurement is done by dipping the pH meter electrode
indicator into the sample.
Fat Content
Fat levels were analyzed using the Roese-Gottlieb
method. Weighing 5 grams of sample, wetted with NH4OH,
then shaken with Polyethylene solvent. The fatty extract in
Polyethylene is collected in a saucer, and the solvent
Polyethylene is evaporated. Then, the extracted fat is
weighed. The calculation of crude fat content can use the
following formula:
Fat Content (% b/b) = 󰇛󰇜
 
Description:
W1: Sample weight
W2: Tube weight + fat
W3: Tube weight after fat removal
W4 : Weight of residual fat in blanks
Protein Levels
Protein levels were analyzed using the Kjeldahl
method. The destruction stage is carried out by weighing 5
grams and then inserted into a 100 ml Kjeldahl tube, which
is then added with concentrated H2SO4 without samples.
Then, it is destroyed at a temperature of 200 – 250ºC for 2 –
3 hours (until the color is clear green). The destruction tube
is cooled, and the volume is adjusted with aquades to 50 ml.
A total of 20 ml is taken and put into a distillation flask, and
then 20 ml of NaOH is added. PP indicator (Phenolphthalein)
is added drop by drop. Samples and blanks were distilled
using 0.2 M HCl solution. The titration process is ended until
the color of the solution in the Erlenmeyer changes from
clear green to clear purple, indicating that all N elements
react with Cl. The calculation of crude protein content can
use the following formula:
Protein Levels (% b/b) =
󰇛 󰇜

Description :
Va: ml HCl sample
Vb: ml HCl titration stamp
N: Normality of standard HCl used
14.007: Atomic weight of nitrogen
6.25: Fruit protein conversion factor
W: Sample weight
Results and Discussion
The Effect of Adding Avocados To Lactic Acid Bacteria
(LAB)
Total LAB testing was conducted to determine the
effect of adding avocados on the growth of lactic acid
bacteria in yogurt drinks. This test uses the Total Plate Count
(TPC) method to see the total amount of BAL that can grow.
The media used is de Man Rogose and Sharpe (MRS) Agar
with the addition of CaCO3 and NaCl solvent. The principle
of the Total Plate Count (TPC) method is to grow microbial

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495
cells that are still alive on a medium so that the microbial
cells multiply to form colonies that can be seen directly by
the eye. Colonies that create a clear zone in the media react
between lactic acid produced by LAB and CaCO3 during
incubation and produce Ca-lactate. The reaction occurs as
follows:
C3H6O3 + CaCO3 Ca(C3H6O3) + H2O + CO2
Figure 1. Growing LAB Colonies
Figure 1. indicates that white spherical colonies are
formed on MRS Agar and CACO3 media. In addition, a clear
zone formed around the colony caused by the reaction
between the acid produced by LAB with CaCO3 and the
solvent NaCl. The total LAB calculation results are shown in
Table 1.
Table 1. The Effect Of Adding Avocados on Lactic Acid
Bacteria (LAB) Yoghurt Drinks
No
Addition of Avocados
(concentration)
Total LAB
(log CFU/mL)
1
0%
9.3x108a ±0.04
2
10%
4.1x109a±0.20
3
15%
4.4x109b±0.07
4
20%
10.53x108a±0.05
Description: values followed by different letters show a
significant difference (p<0.05).
The statistical analysis results using One Way Anova
showed a significant value of p<0.05, indicating the effect of
adding avocados on LAB growth. Furthermore, Duncan's
Post Hoc analysis was carried out to identify significant
differences in each treatment. Statistical results showed a
significant difference at concentrations of 10 – 15%, but at
concentrations of 0%, 10%, and 20%, there was no
noticeable difference. The addition of avocados affects the
growth of LAB yogurt drinks, with optimal growth of LAB
found at a concentration of 15% from 4.1x109a CFU/mL to
4.4x109b CFU/mL.
According to the Indonesian National Standard
(INS), the total LAB in yogurt is at least 107 CFU/mL. An
increase in LAB at a concentration of 15% indicates the peak
of the exponential phase of LAB growth. LAB can grow
rapidly in the exponential phase because it can utilize
nutrients in the growing medium [13].
This follows [14], which states that lactic acid
bacterial cells can grow and divide exponentially to the
maximum number affected by environmental conditions and
nutrients in the media. LAB growth in food is closely related
to the growing medium available. The development of LAB
through the fermentation process can improve the quality of
fermented materials because LAB can produce enzymes that
can degrade fiber [15].
The process of making yogurt is done by
fermentation. Fermentation is a chemical change in food
caused by enzymes. Enzymes that play a role can be
produced by microorganisms or enzymes that already exist
in food [16]. Therefore, adding avocados to yogurt drinks
can increase LAB and lower pH. According to research [17],
the lower the pH and the higher the LAB, the more LAB
utilizes fiber for cell metabolism, hydrolyzes it into simpler
compounds, and ferments it through glycolysis to acids. In
addition, the fermentation process that breaks down milk
lactose into lactic acid can increase acidity, decreasing pH.
The Effect of Adding Avocados on pH
pH testing is carried out to determine the degree of
acidity in the sample using the digital pH meter method. The
data obtained was normal, so a statistical analysis of One
Way ANOVA was carried out. The results showed a
significance value of p<0.05, indicating the effect of adding
avocados to the pH value. Next, Duncan's Post Hoc test was
carried out to see the difference between treatments. The pH
test results are shown in Table 2.
Table 2. The Effect of Adding Avocados on The pH of
Yoghurt Drinks
No
Addition of Avocados
(concentration)
pH
1
0%
4.30a ±0.02
2
10%
4.23b±0.01
3
15%
4.19c±0.01
4
20%
3.89d±0.01
Description: values followed by different letters show a
significant difference (p<0.05).
The results showed a significant difference in the 0 -
20% concentration when adding avocados. The addition of
avocados affects the lowering of the pH value in yogurt
drinks, as seen in table 2. The pH continued to decrease with
each treatment, reaching 3.89 at a concentration of 20%
compared to the initial pH at a 0% concentration of 4.31.
This is due to the age of the starter and the bacteria in it
reaching the logarithmic phase, thus lowering the pH of
avocado yogurt. According to INS, yogurt quality
requirements range from 3.8 – 4.4 pH yoghurt. The decrease
in pH is one of the consequences of the fermentation process
due to the production of lactic acid derived from LAB. This
is in accordance with [18] that the acidity and pH values are
closely related to the increase in metabolism so that lactic
acid production increases while the pH value decreases.
The Effect of Adding Avocados on Fat Content
Fat content testing is carried out to determine the
crude fat content in the sample using the Roese-Gottlieb
method. Fat content is a very important percentage of
nutrients because it is a source of energy to improve texture
and taste and a source of vitamins A, D, E, and K. The
principle of the Roese-Gottlieb method is to extract samples
with diethyl ether and petroleum ether. The fat content test
results are shown in Table 3.

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496
Table 3. The Effect of Adding Avocados on The Fat
Content of Yoghurt Drinks
No
Addition of Avocados
(concentration)
Fat Content
(% b/b)
1
0%
0.05a ±0.007
2
10%
0.33b±0.003
3
15%
0.48c±0.004
4
20%
0.57d±0.005
Description: values followed by different letters show a
significant difference (p<0.05).
The data obtained was normal, so a statistical analysis
of One Way ANOVA was carried out. The results showed a
significance value of p<0.05, indicating the effect of adding
avocados on protein levels. Furthermore, Duncan's Post Hoc
test was carried out to see the significant difference in each
treatment. Statistical results show a significant difference in
concentrations of 0 – 20%. Adding avocados is known to
influence the fat content of yogurt drinks, characterized by
increased concentration. The concentration at the beginning
of adding avocados is 0.05%, which gradually increases to
0.57% after 20%.
Increased fat levels occurred in each treatment with
an average value of 0.05 – 0.57%, according to SNI 01-2981-
1992 [19]. The fat content in yogurt that meets the quality
requirements must have a maximum fat content of 3.8%. The
results showed that the fat content met the INS yoghurt
quality requirements. According to [20], Yogurt fat can be
distinguished from full-fat yogurt (>3%), half-fat yoghurt
(0.5% - 3%), and low-fat yoghurt (<0.5%). Avocado yogurt
is low-fat because avocados have a relatively low-fat content
of 14.66 grams. According to [21], Lactic acid bacteria have
secondary lipolytic activity, meaning lipolytic activity
occurs after other microorganisms break down milk fat into
simpler compounds. During fermentation, the lipase enzyme
hydrolyses fats into simpler compounds, producing fatty
acids and glycerol. The activity of the lipase enzyme reflects
the amount of fat broken down into simpler, more digestible
compounds. Low-fat content goes hand in hand with [22].
Lipotic activity is controlled by lipase enzymes produced by
lactic acid bacteria, and decreasing pH and lipase will free
fatty acids from fat molecules. In addition, fermentation
bacteria also have strong reducing properties. Under active
fermentation conditions, media containing sugar and other
compounds will reduce aldehydes to alcohol, affecting the
increase in fat [23].
The Effect of Adding Avocados on Protein Levels
Protein content testing is done to determine the crude
protein levels in the sample using the Kjeldahl method.
Protein content is the percentage of protein content in a
product, and nasal protein content is the remaining protein
not used by starter bacteria during storage. The principle of
the Kjeldahl method is that nitrogen in proteins is released as
ammonia through a digestion process using concentrated
H2SO4 by heating. Next, ammonia is bound by concentrated
H2SO4 to ammonium sulfate. The results of the protein
content test are shown in Table 4.
The data obtained was normal, so One Way Anova
statistical analysis was conducted. The study's results
showed a significant value of p<0.05, which indicates the
influence of adding avocado on the protein content value.
According to [24], the more concentration of klenceng honey
(Trigona sp) is added, the more yogurt protein levels will
increase. Furthermore, Duncan's Post Hoc test was carried
out to see the significant difference in each treatment.
Statistical results showed a significant difference at
concentrations of 0 – 10%, but there was no significant
difference at concentrations of 10 – 20%. The addition of
avocados has an effect on reducing the protein levels of
yogurt drinks, where protein levels at an initial concentration
of 3.46% decreased to 2.94% at a concentration of 20%.
Table 4. The Effect of Adding Avocados on The Protein
Content of Yoghurt Drinks
No
Addition of Avocados
(concentration)
Protein Levels (% b/b)
1
0%
3.46a ±0.02
2
10%
3.03b±0.08
3
15%
2.98b±0.02
4
20%
2.94b±0.00
Description: values followed by different letters show a
significant difference (p<0.05).
Figure 2. Bale Growth, pH Decrease, and Fat Levels
Increase in Yogurt Based on the Addition of Avocados.
Increased fat levels are also correlated with decreased
protein levels. The decrease in protein levels occurred at a
concentration of 0% from 3.46% to 3.03%; according to the
Indonesian National Standard (INS), good yoghurt has a
minimum protein content of 2.7%. Adding avocados causes
a decrease in protein levels due to increased bacterial
division activity, requiring more amino acids as an energy
source. This follows [25] that optimal BAL formulation and
fermentation time will produce the highest levels of latured
protein. The process of making yogurt using the same lactic
acid bacteria (Lactobacillus bulgaricus and Streptococcus
thermophilus). Lactobacillus bulgaricus bacteria are better
because they ferment glucose and lactose and can grow at 45
- 50 °C temperatures. Lactobacillus bulgaricus is more
tolerant of high acidity [26]. Streptococcus thermophilus is a
spherical type of bacteria [27] with an optimal growth
temperature of 40 - 45 ° C and an acidity tolerance of 0.85 -
0.89%.
According to [28], fat content decreases after
fermentation because Lactobacillus bulgaricus can reduce
fat content by being absorbed as a source for growth. The
9,3
4,1 4,4
10,5
4,31 4,23 4,19 3,89
0,05 0,33 0,48 0,57
3,46 3,03 2,98 2,94
0% 10% 15% 20%
Percentage Extract (%)
Total BAL (CFU/mL) pH
Fat level (% b/b) Protein level (% b/b)

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optimum temperature for the development of Lactobacillus
bulgaricus is 44ºC. These lactic acid bacteria will produce
lipase enzymes, decomposing fat into fatty acids. Then, these
fatty acids will be broken down into compounds with a
distinctive yogurt aroma.
According to [29], the number of living bacterial cells
influences protein levels, and an increase in the number of
living bacterial cells will increase the number of enzymes,
including protein-breaking enzymes (proteases), which are
used to break down proteins (proteolytic activity) and
increase protein synthesis. Proteins will be broken down into
peptides and further hydrolyzed into amino acids. These
breakdown results serve as precursors for enzymatic and
chemical reactions to form flavors.
Conclusion
Based on data obtained in research regarding the
effect of adding avocado (Persea americana) on the
microbiological and chemical quality of yoghurt drinks with
the addition of avocado (Persea americana) concentrations
of 0%, 10%, 15%, 20%. The addition of avocado (Persea
americana) affected the microbiological quality in that the
total BAL value increased from 4.1x109±0.20 CFU/mL
(10%) to 4.4x109±0.07 CFU/mL (15%) so that Avocado
yoghurt drink (Persea americana) is one of the products that
influences LAB growth and reaches optimal levels due to
enzyme activity. The addition of avocado (Persea
americana) affects the chemical quality as the pH value
decreases from 4.30 ± 0.02 (0%) to 3.89 ± 0.01 (20%) so that
the avocado (Persea americana) yoghurt drink. It meets the
quality requirements for the acidity level of yoghurt. The
addition of avocado (Persea americana) affects chemical
quality in that the fat content value increases from
0.05±0.007 % w/w (0%) to 0.57±0.005 % w/w (20%), so it
meets the requirements quality low-fat yoghurt. The addition
of avocado (Persea americana) affects chemical quality. The
protein content value decreases from 3.46 ± 0.02% w/w (0%)
to 2.94 ± 0.00 % w/w (20%), so it is not recommended as
high in protein.
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