Indonesian Journal of Medical Laboratory Science and Technology Open Access
p-ISSN 2684-6748
e-ISSN 2656-9825
RESEARCH ARTICLE
Quality assessment and biochemical characterization of probiotic Lactobacillus species isolated from yogurts fermented either by Tamarindus indica or Capsicum annuum Sowmiya Thiyagarajan 1.
Emmanuel Bhaskar Parthasarathy Mohanalakshmi 2.
Veeraraghavan Gayathri 4.
Santhi Silambanan 1Department of Biochemistry.
Sri Ramachandra Institute of Higher Education and Research.
Chennai.
India 2Department of General Medicine.
Sri Ramachandra Institute of Higher Education and Research.
Chennai.
India 3CEFTE.
Sri Ramachandra Institute of Higher Education and Research.
Chennai.
India 4Department of Biochemistry.
Sri Muthukumaran Medical College and Research Institute.
Chennai.
India Correspondence:
Santhi Silambanan Department of Biochemistry.
Sri Ramachandra Institute of Higher Education and Research (SRIHER) (DU) Chennai.
Tamil Nadu.
India Email: santhisilambanan@gmail.
Article history:
Received: 2024-07-23 Revised: 2024-12-27 Accepted: 2025-01-16 Available online: 2025-04-27 Keywords:
16S rRNA Microbiota Probiotic yogurt Proximate principles https://doi.
org/10.
33086/ijmlst.
Yogurt is used to treat various digestive ailments.
However, there are limited studies of the biochemical and microbiological properties of yogurt.
This study aimed to assess the quality and biochemical characteristics of probiotic Lactobacillus species isolated from yogurts fermented with Tamarindus indica or Capsicum annuum.
Three yogurt samples were used for analysis.
two were homemade, using either Tamarindus indica L.
or Capsicum annuum L.
to initiate Third was a commercial yogurt obtained from the grocery A proximate analysis was carried out.
To identify microbiological strains, yogurt samples were cultured on nutrient- and De Man-Rogosa-Sharpe Biochemical characteristics were examined, and probiotic species were identified through 16S rRNA analysis.
The results showed that traditional yogurts had lower energy content due to reduced carbohydrate and fat levels while exhibiting higher protein content.
` Escherichia coli and coliforms were detected in the commercial yogurt, whereas traditional yogurts were free from these harmful bacteria, highlighting their potential safety.
The biochemical characteristics of all yogurt samples were largely similar.
annuum L.
demonstrated a positive oxidase test, indicating the presence of an electron transport chain in the Lactobacillus species found in the yogurt fermented with annuum L.
Microbial analysis revealed that L.
acidophilus was predominant in traditional yogurt, while commercial yogurt contained L.
These findings suggest that homemade yogurts, with their higher protein content, lower fat and carbohydrate levels, and absence of harmful bacteria, offer a safer and potentially more beneficial probiotic alternative to commercial yogurt.
The specific Lactobacillus strains present in traditional yogurts may contribute to their antimicrobial properties, supporting their probiotic potential.
INTRODUCTION
Yogurt, a key component of Asian culinary traditions for centuries, particularly in regions like the Central Asia, the Middle East, and parts of South Asia .
has a history that can be traced back thousands of years.
Its origins are believed to date to ancient times, when nomadic cultures played a crucial role in discovery of the natural fermentation process of milk, leading to the creation of yogurt.
Yogurt is commonly made from the milk of cow, sheep, buffalo, goat, camel, and other domestic animals .
Initially, the use of Lactobacillus species in fermenting milk was primarily for preservation purposes, thus extending the shelf life of dairy products in the absence of modern refrigeration methods.
This process was observed and refined over generations, leading to the deliberate creation of yogurt as a distinct food item .
Citation: Thiyagarajan S.
Bhaskar E.
Gayathri V.
Mohanalakshmi P.
Silambanan S.
Quality assessment of probiotic Lactobacillus species isolated from yogurts fermented either by Tamarindus indica or Capsicum annuum.
Indones J Med Lab Sci Technol.
:60Ae69.
https://doi.
org/10.
33086/ijmlst.
This is an open access article distributed under the Creative Commons Attribution-ShareAlike 4.
0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly A2025 The Author.
https://journal2.
id/index.
php/IJMLST Sowmiya Thiyagarajan, et al.
Indones J Med Lab Sci Technol.
April 2025.
:60-69
In recent years, understanding Lactobacillus and its role in promoting health benefits has evolved significantly.
While historically, it was used for fermentation and preservation.
Modern research has identified specific strains of Lactobacillus as probiotics.
Probiotics are living microorganisms that offer health benefits when consumed in adequate amounts and should contain at least 106 CFU/mL viable microbial load, according to the International Scientific Association for Probiotics and Prebiotics (ISAPP) .
This transition from merely a fermentation agent to a recognized probiotic illustrates a profound shift in our understanding of how certain microorganisms positively impact human health.
In Turkey, there is a unique variety of yogurt known as cone yogurt, which is distinguished by its fermentation process that involves solely the addition of pine cones to milk, without using of any cultures .
This particular yogurt has a longstanding tradition and has been crafted for numerous years, typically by a select group of producers situated in specific mountain villages within Turkey's Kutahya and Eskisehir regions .
For generations, our ancestors crafted homemade yogurt made from Tamarindus indica L.
and Capsicum annuum L.
, rich in probiotics, unaware of its remarkable health benefits .
Probiotic yogurts which can either by easily prepared at home without expensive starter cultures or commercially procured, and these traditional practices harbored hidden health advantages.
The studyAos novelty was that altered gut microbiota has been found to have significant pathogenesis in various disease states such as diabetes mellitus, major depressive disorder, obesity, nonalcoholic fatty liver disease, and insulin Balancing microbiota with prebiotics and probiotics is essential in handling treatment-resistant cases .
This research aimed to unveil the benefits by isolating and identifying Lactobacillus strains in homemade traditional probiotic yogurt, providing a foundation for future investigations on their health effects.
MATERIALS AND METHODS
In this study, three yogurt samples were used for analysis.
Homemade samples were prepared daily at home.
Fermentation was initiated using either T.
indica L.
or C.
annuum L.
to prepare homemade yogurt samples.
Commercial yogurt samples were freshly purchased daily from the same public grocery shop.
Proximate analysis was conducted on all yogurt samples, including the energy value, carbohydrate, total fat, protein, moisture, ash, fiber, total solids, and titratable The microbiological strains were examined by culturing the yogurt samples in nutrient agar and De Man-RogosaSharpe agar (HiMedia Laboratories Private Lt.
All three samples underwent biochemical characterization of the cultures Probiotic species present in the yogurt samples were identified through 16S rRNA analysis.
Sampling and Preparation of Traditional Probiotic Yogurt Using Tamarindus indica L.
and Capsicum annuum L.
In the southern states of India, homemade traditional yogurts are prepared from cows' milk using T.
indica L.
and C.
annuum L.
to initiate fermentation .
It is an easy process used since ancient times and passed through generations.
The process involves heat treatment, cooling to an incubation temperature, and then subjected to fermentation.
Milk was boiled for 15 minutes to eliminate to the pathogens as well as to denature the whey protein.
The milk was removed from the burner and left at room temperature .
AC) for the milk to reach a temperature of 40-45AC.
Then.
indica L.
or C.
annuum L.
was introduced into 500 mL milk, stirred gently, and left to ferment for 10-12 hours.
After fermentation, a portion of the yogurt was utilized to form further yogurt.
In order to investigate whether the presence of Lactobacillus species is attributable to the microorganisms present in T.
indica L.
or C.
annuum L.
, this study used this backslopping technique for up to 10 preparations .
The yogurts traditionally produced by the back-slopping method are shown in Figure 1.
There was a remarkable increase in colony-forming units (CFU) with subsequent fermentations.
Also, there were enhancements in texture and thickness with each successive generation.
The traditional yogurt prepared with fermentation of T.
indica or C.
annuum is shown in Figure 2.
Proximate Analysis of Yogurt The energy value, carbohydrate, total fat, protein, moisture, ash, fiber, total solids, and total titratable acidity (TTA) were determined using Association of Official Analytical Chemists (AOAC) International guidelines .
The energy content was determined using a bomb calorimeter by measuring the heat released during combustion.
The carbohydrate content was indirectly calculated by subtracting the sum of protein, moisture, ash, and fat from 100%.
The extraction of fat from yogurt involved the use of Soxhlet extraction with petroleum ether.
The percentage of total fat was calculated based on the weight of extracted fat with the initial weight of the yogurt sample.
The AOAC Kjeldahl method was employed for protein analysis, involving yogurt sample digestion with concentrated sulfuric acid (H 2SO.
to release ammonia (NH.
, subsequently titrated using standardized HCl.
Protein percentage was calculated by determining the nitrogen content obtained from the titration.
Moisture content was determined by oven-drying five-gram yogurt sample at 105AC until a stable weight had reached.
The loss in weight represented the moisture content.
Ash content was obtained by incinerating yogurt samples in a muffle furnace at 550AC until complete combustion of organic matter occurred yielding inorganic ash Fiber content was determined by treating the sample with H2SO4 and NaOH to eliminate non-fibrous elements.
The resulting residues were filtered, dried, and weighed.
Total solids were determined by drying 10 grams of yogurt sample Sowmiya Thiyagarajan, et al.
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until a constant weight was achieved.
The TTA is a measure of the amount of lactic acid in the yogurt, and was expressed as a percentage.
TTA was determined by titrating 10 grams of yogurt sample with standardized NaOH and phenolphthalein The amount of NaOH consumed indicated the acidity.
The samples were also tested for their pH and sensory characteristics .
Safety and Quality Profile of Traditional Yogurts and Commercial Yogurt The quality of yogurt is based on its palatability and sensory properties, such as texture, color, and appearance.
Therefore, establishing tolerance limits for these attributes is essential in yogurt production to ensure consistent product The typical sensory characteristics of yogurt include creamy or white color, soft and smooth semi-solid structure, spreadable or spoonable consistency, and slightly acidic, sour taste.
These properties are influenced by protein content, fermentation processes, and texture .
As much as 0.
1 mL of each yogurt sample was inoculated to the agar surface using an L-rod.
The plates were incubated for 24-48 hours at 37AC.
Isolated colonies were sub-cultured on freshly prepared MRS agar plates to obtain a pure Lactobacillus culture.
Colony characteristics, colony count.
Gram staining, and microscopic morphological features of the isolates were analyzed.
Both traditional yogurt samples and commercial yogurt sample were examined for the presence of common pathogenic microorganisms.
Selective media, including membrane Filter Fecal Coliform Agar .
FC) and MacConkey agar (MAC), were used to identify Escherichia coli and coliforms, while Sabouraud Dextrose Agar (SDA) was employed to identify fungi and yeasts.
These media were specifically formulated to promote the growth of E.
coli, coliform bacteria, and fungi .
Biochemical Characteristics of Microbial Isolates The microbial isolates from the three yogurt samples were tested for biochemical characteristics based on the method by Rahman et al .
Indole test The tryptone broth .
grams of Tryptone dissolved in 1000 mL of distilled wate.
was inoculated with the test This mixture was incubated at 37AC for 24-48 hours.
After incubation, approximately 0.
3 mL of Kovac's reagent .
grams para-dimethyl amino Benzaldehyde, 75 mL butyl alcohol, and 25 mL concentrated HC.
, was added.
The mixture was thoroughly mixed and left to stand for 30 minutes.
The formation of a brown ring on the broth's surface indicated the presence of indole production .
Heated whole raw milk to 85CC for 20-30 minutes Cooled milk to 40-45CC Introduced Tamarindus indica L.
/ Capsicum annuum L.
into cooled milk and stirred well Kept aside for fermentation .
-12 hour.
Yogurt was obtained Back slopping method -retained some yogurt for subsequent preparations Used retained yogurt from each preparation for 10 subsequent preparations There was consistent increase in CFU, texture and thickness with subsequent generations Figure 1.
Preparation of traditional probiotic yogurt using Tamarindus indica L.
and Capsicum annuum L.
Figure 2.
Traditional probiotic yogurt prepared with fermentation of Tamarindus indica L.
or Capsicum annuum L.
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Methyl red test The Methyl red-Voges Proskauer (MR-VP) broth .
g/L buffered peptone, 5 g/L dextrose, 5 g/L K2HPO4, 5 g/L NaCl, and adjusted to a pH of 7.
, was inoculated with the culture and incubated at 37AC for 24-48 hours.
The appearance of red color with the addition of methyl red reagent was considered positive.
The color change denoted the presence of microorganisms participating in the mixed-acid fermentation pathway .
Voges-Proskauer test The MR-VP medium inoculated with the culture was incubated at 37AC for 24-48 hours.
After incubation, 3 mL of Barrit's reagent A .
% alpha naphthol in 95 mL of absolute ethano.
and 1 mL of Barrit's reagent B .
g/L KOH, 3g/L) were added.
The tubes were agitated and then left undisturbed for 15 minutes.
The appearance of red color indicated a positive test .
Urease test The urease agar medium .
g/L dextrose, 1.
5 g/L peptic digest of animal tissue, 5 g/L NaCl, 2 g/L KH2PO4, 0.
phenol red, 15 g/L agar, with a final pH of 6.
8A0.
2 at 25AC), was inoculated with the culture and incubated for 24 hours at 37AC.
During this incubation period, the breakdown of urea led to the production of NH 3, causing the medium to become alkaline and exhibit a pink-red coloration .
Triple sugar iron test (TSI) The TSI agar medium .
g/L beef extract, 20 g/L peptone, 3 g/L yeast extract, 10 g/L lactose, 10 g/L sucrose, 1 g/L dextrose monohydrate, 0.
2 g/L FeSO4, 5 g/L NaCl, 0.
3 g/L NaCCSCCOCE, 0.
024 g/L phenol red, 12 g/L agar, adjusted to a pH of .
was conducted to assess the microorganism's ability to ferment sugars and produce hydrogen sulfide (H 2S).
The acidic byproducts of fermentation resulted in a yellow color, while alkaline byproducts yielded a red color.
The formation of H2S was indicated by a black color .
Citrate utilization test The Citrate Agar .
g/L NaCl, 0.
2 g/L MgSO4,1 g/L NHCEHCCPOCE, 1 g/L K2HPO4, 2 g/L sodium citrate, 0.
08 g/L bromothymol blue, 15 g/L agar .
at a pH of 6.
The citrate utilization test was used to detect the ability of an organism to utilize citrate as the sole carbon source for its growth .
Catalase test A small amount of culture was placed over a clean slide.
A drop of 3% hydrogen peroxide was placed over the culture and observed for effervescence.
The effervescence production showed the organism's ability to produce the enzyme, catalase .
Oxidase test Cytochrome oxidase in mitochondria and aerobic bacteria function to reduce molecular oxygen to yield water and generate energy by the formation of a transmembrane electrochemical gradient that drives ATP synthesis and other energy-demanding processes in the cells.
Thus, the oxidase test was conducted to determine the presence of cytochrome c oxidase, an enzyme involved in the electron transport chain of certain bacteria.
A small amount of culture was placed over a clean slide.
Then a drop of oxidase reagent was added, and a purple or dark-blue color change developed within 10-30 This indicated a positive result .
resence of oxidas.
A lack of color change or a light pink coloration was considered a negative result .
Carbohydrate fermentation test A carbohydrate fermentation medium .
g/L peptone, 1 g/L beef extract, 5 g/L NaCl, 0.
018 g/L phenol re.
were dispensed 5 to 7 mL .
r the volume required to submerge the Durham tub.
of the broth into test tubes.
It was ensured that each Durham tube was fully submerged without any trapped air bubbles.
Then, the caps or cotton plugs were loosely secured and autoclaved the tubes for 15 minutes at 121AC and 15 lb pressure.
Gas production along, with pH decrease were shown by a color change in the medium, which indicated positive carbohydrate fermentation.
If there was no gas production or pH change, it suggested negative fermentation of the tested carbohydrate by the microorganism .
Isolation and 16S rRNA Identification of Bacterial Isolates DNA was isolated for 16S rRNA sequencing, as this gene was a highly conserved region found in all the bacteria and archaea genomes.
By isolating the DNA, it was ensured that we were working with the complete genetic material of an organism, allowing for the amplification, sequencing, and detailed analysis of the 16S rRNA gene.
Sequencing this gene Sowmiya Thiyagarajan, et al.
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allowed for the identification and classification of microorganisms, microbial diversity analysis for taxonomic identification, and phylogenetic analysis .
The isolates were grown on MRS broth till they reached the log phase .
hours-old culture.
The cells were centrifuged at 5,000xg for 15 minutes.
Extraction of DNA was performed using the HiMedia MB505 (HiPurA Bacterial Genomic DNA Purificatio.
In 200 AL of lysozyme solution, a loopful of the culture was suspended .
mg/mL, i.
, 2.
x 106 units/mL), and the suspension was incubated at 37CC for 30 minutes.
RNase A solution .
mg/mL) was added and incubated at room temperature for two minutes.
20AL of Proteinase K solution .
mg/mL) was added, followed by 200AL of the lysis solution, vertexing the entire mixture, and incubated for ten minutes at 55AC.
DNA was precipitated by the addition of 200AL of chilled 100% ethanol.
The mixture was vortexed and the lysate was loaded onto the HiElute Miniprep Spin Column (HiMedi.
as well as centrifugation was done at 6,500xg for three minutes.
The flow-through liquid was discarded and 500AL of prewash solution was loaded into the spin column.
This step was repeated three times.
Lastly, the spin column was centrifuged for one minute at a maximum speed of 10,000xg to dry the column.
The spin column was positioned on a fresh collection tube and 200AL of Elution Buffer was carefully loaded directly into column.
For five minutes, the column was incubated at room temperature.
The column was centrifuged at 6,500xg for 2 minutes to extract the DNA.
A UV spectrophotometer was employed to determine the DNA concentration (Shimadzu Corporation A11454806.
The isolated DNA was amplified for the 16S rRNA region by Polymerase Chain Reaction (PCR) utilizing a Biometra thermal cycler (T-Personal .
A PCR reaction mix .
rocured from GeNe.
consisting of 2.
5AL of 10X buffer, 1AL of primers, 2.
5AL of 2.
5mM of each dNTP, 1AL template DNA as well as 2.
5 U of Taq DNA polymerase, and nuclease-free water was prepared.
PCR amplification cycle was as follows: one cycle of 5 minutes at 94CC, 35 cycles of 1 minute at 94CC, cooling for 1 minute at 50CC, two minutes at 72CC, as well as an additional cycle of 7 minutes at 72CC.
PCR was conducted using specific forward and reverse primers targeting the 16S rRNA gene .
This amplification step facilitated the generation of multiple copies of the 16S rRNA gene, preparing the DNA for sequencing .
The amplified PCR product was loaded onto a 1% agarose (HiMedi.
gel to confirm successful amplification.
Electrophoresis was carried out using Tris-Acetate-EDTA (TAE) buffer, and DNA bands were visualized with ethidium bromide staining.
The PCR product was purified using manufactured protocol from the AxyPrep PCR Clean-up Kit (Axygen.
AP-PCR-.
Sequence Similarity and Phylogenetic Analysis The purified DNA was used for sequencing (Applied Biosystems 3730xl DNA Analyzer.
USA), along the chromatogram obtained was analyzed.
The NCBIAos (National Center for Biotechnology Informatio.
nucleotide BLAST (Basic Local Alignment Search Too.
capability was employed to evaluate the DNA sequences.
The consensus sequence was compared for similarity in the NCBI GenBank database using BLAST analysis .
The Maximum Likelihood method was employed alongside the Kimura 2-parameter model to understand the evolutionary relationships among the analyzed taxa .
The percentage of times the associated taxa clustered together in the bootstrap test .
,000 replicate.
was indicated beside the branches.
It depicted the evolutionary links among the taxa.
Branches supported by less than 50% of bootstrap replicates were condensed .
Initial trees were generated using Neighbor-Join and BioNJ algorithms applied to pairwise distance data estimated through the Maximum Composite Likelihood (MCL) approach to initiate the analysis.
The topology with the highest log likelihood value was selected.
This analysis involved 11 nucleotide sequences, encompassing codon positions first, second, third, and noncoding regions.
Positions containing gaps or missing data were excluded, resulting in a final dataset of 1434 The evolutionary analyses were conducted using MEGA7 software.
MEGA is a molecular evolutionary genetics analysis (MEGA) software program were used to generate phylogenetic trees.
Bootstrap analysis was a method used to estimate the statistical reliability of clades in phylogenetic trees.
The bootstrap value was the proportion of replicate phylogenies that recovered a particular clade from the original phylogeny .
RESULTS AND DISCUSSION
Proximate Analysis of Traditional Yogurts and Commercial Yogurt In the present study, both the traditional yogurts exhibited reduced energy, carbohydrate, and fat content compared to commercial yogurt.
This is indicating that indicating a potentially healthier nutritional profile for calorie-conscious In terms of protein content, both traditional yogurts exhibited higher levels, while commercial yogurt had lower protein levels.
Moisture and ash content were similar among the traditional yogurt samples, while commercial yogurt exhibited higher ash content.
Fiber content was negligible across all the samples.
Table 1 indicated that all yogurt types contain very low fiber, below 0.
2 grams/100 grams.
Total solids were present at concentrations of 55.
95% and 52.
95% in indica L.
derived traditional yogurt and C.
annuum L.
derived traditional yogurt, while commercial yogurt demonstarted a level of 52.
62% (Table .
The traditional yogurt samples yielded lesser energy, and contained lesser carbohydrates, and total fat but more proteins than commercial yogurt which are beneficial for obese as well as diabetic individuals.
Other parameters such as moisture, fiber, and total solids were the same among the three samples.
Titratable acidity was higher in commercial yogurt Sowmiya Thiyagarajan, et al.
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compared to traditional yogurts.
This results were in alignment with study by Katidi et al.
which showed that the carbohydrate, fat and protein contents were 1Ae12 grams/100 grams, 0Ae20 grams/100 grams and 3.
3Ae11 grams/100 grams.
According to Saeed .
energy content was 85.
09Ae104.
75 Kcal/100 grams, moisture was 79-80%, ash content was 0.
28Ae0.
95% and titrable acidity was 0.
Kowaleski et al.
showed that increasing milk total solids from 16 to 23 grams/100 grams improved the growth of Lactobacillus bulgaricus where counts of this organism after 240 minutes in 23% total solids is the highest.
Safety and Quality Profile of Traditional Yogurts and Commercial Yogurt Regarding sensory characteristics, all the yogurt samples exhibited white color, smooth appearance, thick and creamy texture, and a subtle combination of sour and sweet taste, with a pH range of 4-5.
The variations in yogurt color were attributed to the different biochemical activities of yogurt microorganisms, which transformed the casein complex from a micellar to a dispersed state.
The consistency corresponded to textural and rheological measurements .
The yogurt samples complied with Food and Drug Administration specifications, which stated that yoghurt should have a maximum pH of 4.
The low pH value was the result of the amount of lactic acid produced from the fermentation of milk lactose by bacteria.
Due to the low pH value, calcium would be converted to its ionic form, making it highly bioavailable for intestinal absorption.
Also, the inhibitory effect of dietary phytic acid on calcium bioavailability would be reduced by the low pH value.
The low pH value of yogurts also inhibited the growth of pathogens present in the yoghurts .
However, when assessing the microbial safety.
Coliforms such as E.
coli was detected in the commercial yogurt, while both traditional yogurts showed the absence of these harmful bacteria.
Fungi was absent in all the samples (Table .
The safety and quality profiles of all the three samples showed the same characteristics.
However.
coli and coliforms were present in the commercial yogurt sample.
Poor hand washing, improper cleaning of equipment, and contaminated surfaces can contribute to introduction of E.
coli and coliforms.
coli in yoghurt brands is a serious health concern, as it can have negative implications for consumers.
This poses a serious health risk to consumers, as these bacteria can cause severe infections like food poisoning and septicemia.
The isolated E.
coli resisted various antimicrobial agents, indicating a potential public health concern.
The study highlighted the importance of proper hygiene and sanitation in yoghurt production to prevent contamination with harmful pathogens like E.
This highlighted the need for improved quality control and sanitation measures in yogurt production to ensure consumer safety .
The absence of Coliforms in the traditional yogurt samples could be due to specific strains of Lactobacillus species present in T.
indica L.
and C.
annuum L.
These Lactobacillus strains exert antimicrobial properties, thus, effectively suppressing the growth of pathogenic bacteria.
This competitive inhibition or antimicrobial activity of the Lactobacillus strains could be one of the key reasons behind the absence of E.
coli and coliforms in the traditional yogurt samples .
The findings highlighted the potential superiority of traditional yogurts, particularly in terms of microbial safety, making them a preferable choice for consumers.
Biochemical Characteristics of Microbial Isolates from Yogurt Samples Regarding their general characteristics, all three types of isolates grown on MRS agar exhibited tiny and round growth with a creamy, white-colored surface.
Gram staining for all isolates showed a positive result, and the colony-forming units per milliliter (CFU/mL) were too numerous to count (TNTC), indicating abundant growth.
The Indole test.
Citrate utilization test.
Methyl red test.
Urease test.
Triple Sugar Iron test.
Voges-Proskauer test.
Catalase test, and Oxidase test were all negative across all the three types of yogurts, except for the oxidase test, which showed a positive result in the C.
annuum L.
traditional yogurt isolate.
Glucose and mannitol fermentation were positive in all the types of yogurts.
Lactobacillus bacteria converted glucose and other carbohydrates into lactic acid through fermentation.
This indicated that Lactobacillus species was present in the sample.
Sucrose and fructose fermentation were positive in T.
indica L.
and C.
annuum L.
traditional yogurts, while commercial yogurt showed mixed results with sucrose fermentation.
Maltose fermentation was observed only in the commercial yogurt isolate (Table .
Table 1.
Proximate analysis of traditional yogurts and commercial yogurt indica L.
Parameters Traditional yogurt Energy (Kcal/100 gram.
Carbohydrate .
rams/100 gram.
Total fat .
rams/100 gram.
Protein .
rams/100 gram.
Moisture (%) Ash (%) Fiber .
rams/100 gram.
<0.
Total solids (%) Titratable acidity (%) annuum L.
Traditional yogurt <0.
Commercial <0.
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Table 2.
Safety and quality profile of traditional yogurts and commercial yogurt samples indica L.
annuum L.
Parameters Traditional Yogurt Traditional Yogurt Sensory characteristics Color White White Appearance Smooth Smooth Texture Thick, creamy Thick, creamy Taste Subtle sour, sweet Subtle sour, sweet Absent Absent Coliform Absent Absent Fungi Absent Absent Table 3.
Biochemical characteristics of microbial isolates from yogurt samples indica L.
annuum L.
Parameters Traditional Yogurt
Traditional Yogurt
General Characteristics MRS agar Tiny Tiny Color
White
White
Surface Creamy Creamy Gram staining CFU/mL TNTC
TNTC
Biochemical Characteristics Indole test
Methyl red test
Voges-Proskauer test
Urease test
Triple sugar iron test
Citrate utilization test
Catalase test
Oxidase test
Carbohydrate fermentation Glucose Sucrose Fructose Maltose Mannitol
Expected Lactobacillus
Lactobacillus
Microorganisms Commercial Yogurt
White
Smooth
Thick, creamy Subtle sour, sweet
Present
Present
Absent
Commercial Yogurt
Tiny, round
White
Smooth
TNTC
/ /
Lactobacillus species : Answers the test.
-: Do not answer the test.
* TNTC: too-numerous-to-count The biochemical characteristics were almost the same for all the yogurt samples.
annulus L.
showed a positive oxidase test, which indicated the presence of a functioning electron transport chain in the Lactobacillus species present in yogurt samples fermented by C.
annulus L.
Glucose, sucrose, fructose and mannitol except maltose were fermented by the home-made yogurt samples.
On the contrary.
Lactobacillus species were present in commercial yogurt fermented glucose, maltose, and mannitol, while ambiguous results were obtained for sucrose and fructose.
The fermentation test helped to identify the bacteria.
These findings suggested both similarities and differences among the microbial isolates from these yogurt samples in terms of their biochemical characteristics and carbohydrate fermentation, potentially indicating distinct microbial compositions in these yogurt types.
Authentication and Identification of Lactobacillus species Isolates Identifying of Lactobacillus at species and strain level involved a combination of physiological and biochemical characteristics, as well as molecular techniques for accurate authentication.
The microbial isolates obtained from yogurt samples were identified using the 16S rRNA gene sequencing technique, and amplified PCR products were purified and Similarity sequence search for selected Lactobacillus isolates (T.
indica L.
traditional yogurt isolate.
traditional yogurt isolate.
Commercial yogurt isolat.
(NCBI.
BLAST) revealed 99% homology with the available sequence of L.
acidophilus and L.
The phylogenetic trees, which are graphical representations of the evolutionary relationships among different species or genes, in Figure 3 illustrate the relatedness of these isolates to the sequences retrieved from the database.
Sample 1 illustrated that the L.
acidophilus isolates closely clustered with reference strains NR 1.
NR 117062.
NR 113638.
1, and NR 043182.
1, all showing robust 100% bootstrap support.
This close clustering strongly suggested that the isolate shared a high degree of similarity with the established L.
acidophilus strain.
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Sample 1 Sample 2 Sample 3 Figure 3.
Molecular Phylogenetic analysis of Lactobacillus species from T.
indica L.
and C.
annuum L.
(Sample .
traditional and commercial (Sample .
yogurt microbial isolates by Maximum Likelihood Method.
Therefore, the T.
indica L.
traditional yogurt isolate was confirmed to contain L.
Sample 2, the L.
acidophilus isolates formed a tight cluster with reference strains NR 117062.
NR 113638.
NR 117812.
1, and NR 1 with 100% bootstrap support.
This close grouping indicated significant similarity between the isolated strain and known reference strains.
Thus, the C.
annuum L.
traditional yogurt isolate was identified to have L.
Sample 3 demonstrated that the L.
plantarum isolates clustered closely with reference strains NR 112690.
NR 115605.
1, and NR 117813.
1, showing a strong 96% bootstrap support.
This clustering strongly suggested a high resemblance between these strains thus the commercial yogurt isolate was found to be L.
plantarum strains.
Dairy products are widely recognized as effective carriers for introducing beneficial probiotic bacteria into the human digestive system.
Presently, there is a significant market demand for probiotic products.
During the fermentation process, lactose in yogurt gets broken down, making it easier for lactose-intolerant individuals to consume without experiencing diarrhea, flatulence, and other symptoms associated with this condition.
The breakdown of lactose into lactic acid during fermentation reduces the digestive challenges for those who have difficulty digesting lactose, enabling them to enjoy yogurt without experiencing adverse effects .
In contrast to homemade or traditional yogurts, commercial yogurt often contains food additives and preservatives that can have diverse consequences, depending on the additives used.
Food additives like maltodextrin and some noncaloric artificial sweeteners present in commercial yogurt have shown harmful effects on gut health.
Studies indicate that certain artificial sweeteners, such as saccharin, can lead to dysbiosis in the gut microbiota, potentially contributing to glucose intolerance .
These additives compromise the health benefits of probiotics, which are the primary reason for consuming commercial probiotic yogurt.
In contrast, homemade traditional yogurt with probiotics appears to be CONCLUSIONS The traditional yogurt samples yielded lesser energy and contained lesser carbohydrate and total fat but more proteins than the commercial yogurt, which is beneficial for obese as well as diabetic individuals.
The safety and quality profiles of Sowmiya Thiyagarajan, et al.
Indones J Med Lab Sci Technol.
April 2025.
:60-69
all the three samples showed the same characteristics.
However.
coli and coliforms were present in the commercial yogurt The biochemical characteristics were almost the same for all the yogurt samples.
annulus L.
showed a positive oxidase test, which indicated the presence of a functioning electron transport chain in the Lactobacillus species present in yogurt samples fermented by C.
annulus L.
Glucose, sucrose, fructose, and mannitol were fermented by the home-made yogurt samples except for maltose.
The fermentation test helped to identify the bacteria.
The specific strains of Lactobacillus species present in both traditional yogurts contributed to their antimicrobial properties.
In summary, the findings suggest that traditional yogurts, with their distinctive characteristics and potential health benefits, could be considered the preferable choice for consumers seeking a wholesome and safe yogurt option.
Further, in vivo studies are being planned to elucidate its human health benefits for specific diseases.
Author contributions: ST.
SS: Conceptualization, methodology, validation, resources, data curation, funding acquisition.
SS.
EB: software.
ST.
SS.
EB.
GV.
MP: formal analysis, writingAioriginal draft preparation, writingAireview and editing, visualization, supervision.
SS.
GV: project administration.
All authors have read and agreed to the published version of the Funding: This research was funded by Shri N.
V Ramasamy Udayar Research Fellowship, grant number U02M210724 and the APC was funded by ST .
The funding received was sufficient to conduct the research.
Acknowledgements: Authors wish to thank SRIHER for providing necessary infrastructure to conduct the necessary.
Ethics statement: Since the study did not involve animals or human beings, it did not require ethics approval.
Conflict of interest: None.
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