Indigenous Lactobacillus Isolates from Indian Dairy: Screening, Characterization, and Sensory Evaluation for Quality Dahi Production

Introduction

Dahi is a traditional fermented milk product widely consumed across India and is prepared through natural fermentation of milk by indigenous Lactic acid bacteria. The quality and consistency of traditionally made Dahi depend largely on the microbial composition and fermentation activity of the starter cultures present in milk. However, traditional Dahi production often lacks standardization and control, resulting in variable product quality and inconsistent sensory attributes. The use of well-characterized, pure Lactobacillus cultures as starter strains offers a practical solution to improve the quality, safety, and consistency of Dahi production at both household and commercial scales. Indigenous Lactobacillus strains isolated from traditional Dahi and raw milk represent a valuable resource for starter culture development. Lactobacilli isolated from naturally fermented Dahi possess probiotic characteristics, including acid tolerance and antimicrobial activity against pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and represent viable candidates for use as novel starter cultures in dairy fermentation [1] . These native strains are naturally adapted to milk fermentation conditions and can be easily maintained and propagated by dairy producers at local levels through simple back-slopping techniques. The interest in region-specific ethnic fermented foods and their functional microbiota is rising, with growing demands for functional foods possessing multiple beneficial attributes and low safety concerns [2] .

Many Lactobacillus strains exhibit strong lactose fermentation and acidification capacity, which enables rapid pH reduction, improved safety, and extended shelf-life in dairy products [3] . Additionally, during milk fermentation, enzymes released from Lactobacillus cells decompose milk proteins and fats, contributing to desirable changes in texture, flavor, and overall quality of fermented dairy products [4] . The selection of appropriate Lactobacillus strains with good fermentation characteristics is therefore essential for producing high-quality Dahi with improved organoleptic and keeping quality. Furthermore, starter cultures significantly improve the consistency and shelf-life of fermented dairy products such as yogurt, cheese, and traditional preparations, while simultaneously offering organoleptic improvements in flavor and aroma [5] . The objective of the present study was to isolate and identify Lactobacillus strains from Dahi and raw milk samples collected from Chhattisgarh. Isolates were characterized based on morphological features, Gram reaction, catalase activity, and carbohydrate fermentation patterns using the HiCarbo KB009A sugar utilization kit. On the basis of their fermentation profiles, selected isolates were then used as pure starter cultures to prepare Dahi and evaluate their performance in milk fermentation.

Material and Method Isolation of Lactobacillus Isolates

Lactobacillus strains were isolated from Dahi and raw milk samples collected from dairy sources in Chhattisgarh, India. Samples were serially diluted in sterile physiological saline (0.85% NaCl) up to 10⁻⁸ dilution. Isolation was performed using de Man, Rogosa, and Sharpe (MRS) agar (HiMedia Laboratories, Mumbai, India) by both pour plate and streak plate methods. Pour plates were prepared by mixing 1 mL of appropriate dilutions with molten MRS agar and incubating at 37 °C for 48 hours under anaerobic conditions. Streak plate isolation was carried out by streaking diluted samples onto MRS agar plates and incubating under the same conditions. Colonies with typical translucent to whitish appearance characteristic of lactobacilli were selected and transferred to MRS broth (HiMedia Laboratories) for purification. Pure cultures were maintained in MRS broth at 4 °C for short-term storage.

Morphological Characterization

Morphological identification of isolates was performed using Gram staining. Smears were prepared from overnight MRS broth cultures, heat-fixed, and stained following standard Gram staining protocol. Microscopic examination was conducted under an oil immersion objective lens (100× magnification) using a light microscope. Gram-positive rod morphology characteristic of Lactobacillus species was observed and recorded. Catalase activity was determined by adding 3% hydrogen peroxide to colonies on MRS agar plates and observing for bubble formation; lactobacilli typically show catalase-negative or weakly catalase-positive reactions.

Evaluation of Fermentation Intensity and Acidification Capacity

During HiCarbo KB009A incubation at 37 °C, fermentation reactions were recorded based on color change in the twelve carbohydrate wells: lactose, xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melibiose, sucrose, L-arabinose, and mannose. The kit uses a pH-sensitive indicator system where positive fermentation is indicated by red-to-yellow color shift, while red coloration indicates negative fermentation. The intensity and speed of red-toyellow color change correlate directly with acid production rate and metabolic robustness [6] . The stringent selection based on rapid fermentation in lactose and dextrose, the primary milk carbohydrates, ensured only isolates with robust, predictable acidification capacity were retained. This protocol guaranteed that selected isolates would deliver consistent curd formation, predictable fermentation duration, and desirable sensory properties in Dahi production without fermentation defects such as bitterness or graininess.

Preparation of Dahi

Prior to Dahi preparation, the five selected Lactobacillus isolates were inoculated individually into sterile skim milk test tubes using a sterile inoculating loop under aseptic conditions. The cultures were incubated at 37 °C for 24 hours to allow fermentation and acid development in the skim milk. The fermented skim milk culture was then added at the rate of 2% (v/v) to freshly pasteurized milk (500 mL) held at 45 °C. The inoculated milk was mixed gently and transferred to sterile containers, then incubated at 37 °C until curd formation and whey separation were observed (approximately 4-6 hours). The prepared Dahi samples were cooled to room temperature and stored at 4 °C until sensory evaluation

Sensory Evaluation

Sensory evaluation of Dahi prepared with each Lactobacillus isolate was conducted using a 9-point hedonic scale (9 = extremely liked; 1 = extremely disliked) by a panel of 10 untrained panelists. Samples were evaluated for color, texture (consistency and firmness), and taste (sourness and flavor intensity). All samples were presented at room temperature (approximately 25 °C) in coded containers to avoid bias. Panelists were provided with water for mouth rinsing between samples. Mean scores for each attribute and each isolate were calculated and recorded.

Statistical Analysis

Mean values and standard deviations of sensory scores were calculated for each attribute. Data were analyzed using descriptive statistics to identify the isolate(s) producing Dahi with the most acceptable sensory characteristics.

Result and Discussion Isolation and Identification of Lactobacillus Isolates

A total of 60 samples comprising 30 Dahi and 30 raw milk samples were collected from local dairy shops and dairy farms located in Karnal and nearby regions including Indri, Nilokheri, Uchani, Kurukshetra, and Penwa in Haryana, India. Initial isolation on MRS agar followed by morphological characterization by Gram staining and colony morphology examination revealed 38 presumptive Lactobacillus isolates (63.3% recovery rate) exhibiting characteristic Gram-positive rod morphology and translucent to whitish colony appearance. The high isolation rate reflects the natural abundance of lactobacilli in traditional Dahi and raw milk, confirming these sources as rich reservoirs of indigenous Lactobacillus populations [1] . Following morphological identification, all 38 isolates were subjected to carbohydrate fermentation profiling using the HiCarbo KB009A sugar utilization kit. Among these 38 isolates, 22 isolates (57.9%) exhibited characteristic Lactobacillus fermentation patterns and were retained for further work, while 16 isolates (42.1%) displaying atypical or abnormal sugar fermentation patterns were discarded. The characteristic fermentation profile of retained isolates included rapid lactose and galactose fermentation with variable fermentation of arabinose, xylose, and other carbohydrates, consistent with standard taxonomic criteria for genus Lactobacillus. The retained 22 isolates were propagated in MRS broth, and glycerol stocks (25% v/v glycerol) and slants were prepared and stored at -20 °C and 4 °C respectively for future use and reference.

Selection of Cultures for Dahi Preparation

Among the 22 retained isolates, six isolates (NLK, PWA, SNP, PNT, KD, and KA) demonstrated the most pronounced and rapid color change in the HiCarbo KB009A kit, indicating strong acid production. All six exhibited clear yellow coloration in lactose and dextrose wells within 10 hours at 37 °C, indicating efficient fermentation of primary milk carbohydrates. Additionally, all six showed positive fermentation in maltose, sucrose, and galactose wells, confirming metabolic versatility. Selection based on rapid fermentation patterns is essential for predictable Dahi production. Cui & Qu [7] reported that Lacticaseibacillus casei and Lacticaseibacillus paracasei strains ferment diverse carbohydrates including glucose, lactose, sucrose, and maltose, conferring metabolic flexibility. Barzegar et al., [6] documented that Lactobacillus isolates exhibiting strong carbohydrate fermentation kinetics demonstrate superior acid production, correlating with consistent curd formation and desirable organoleptic properties. The rapid lactose and dextrose fermentation demonstrated by the six selected isolates, combined with galactose and maltose fermentation, establishes their suitability as high-performance starter cultures for standardized Dahi production.

Significance of Indigenous Lactobacillus Populations

The recovery of 38 Lactobacillus-like isolates from 60 samples demonstrates that both Dahi and raw milk collected from the Karnal region contain substantial populations of indigenous lactobacilli with significant potential for dairy fermentation and probiotic applications. Previous studies on lactobacilli isolated from indigenous Indian fermented dairy products have demonstrated that the majority of isolates exhibit high acid tolerance, with survivability rates ranging between 85 to 97% at pH 3.0, indicating good tolerance against acidic conditions of the gastrointestinal tract [6] . These findings underscore the inherent stress-tolerance capacity of indigenous Lactobacillus strains naturally selected through generations of traditional fermentation practices. Strains of Lactobacillus acidophilus isolated from indigenous Dahi have been shown to possess excellent probiotic and functional attributes, including gastrointestinal transit tolerance, strong cell surface hydrophobicity enabling survival and colonization in the gastrointestinal tract, exopolysaccharide production, and antioxidant potential [8] . The recovery of multiple Lactobacillus isolates from a single geographical region (Karnal and surrounding areas) in the present study suggests regional microbial diversity and the potential existence of locally adapted strains with unique fermentation characteristics suited to the specific conditions of milk in this region. The elimination of 16 isolates (42.1%) based on abnormal sugar fermentation patterns was a crucial quality control step. Abnormal fermentation patterns may indicate misidentified genera, mixed cultures, or isolates with atypical metabolic profiles unsuitable for controlled dairy fermentation. The interest in region-specific ethnic fermented foods and their functional microbiota is rising, with demands for functional foods possessing multiple beneficial attributes and low safety concerns driving research toward the isolation and characterization of lactobacilli candidates from locally resourced fermented foods for the development of ready-to-eat fermented functional products [6] . This selection approach ensures that only genuinely characteristic Lactobacillus isolates are retained for downstream technological and sensory applications.

Indigenous Lactobacillus as Stress-Tolerant Starter Cultures

The 22 retained isolates represent a valuable collection of indigenous Lactobacillus strains with demonstrated technological potential. Indigenous dairy lactobacilli are inherently adapted to milk fermentation conditions and possess several advantages over commercial cultures: they survive the acidic and bile-rich conditions of the human gastrointestinal tract, produce antimicrobial compounds, exhibit resilience to temperature fluctuations common in rural dairy environments, and maintain fermentation activity under suboptimal storage conditions. Lactic acid bacteria strains isolated from naturally fermented milk products have demonstrated tolerance to low pH (3.0), bile salts, lysozyme resistance, and β-galactosidase activity, with some strains showing greater than 85% cell surface hydrophobicity, indicating probiotic potential [9] . The preservation of isolates as glycerol stocks and slants ensures long-term viability and maintains the genetic integrity of these cultures for future use. Glycerol at 25% concentration is a standard cryoprotectant that minimizes ice crystal formation and cellular damage during freezing, thereby preserving the physiological and fermentation characteristics of Lactobacillus strains. This approach facilitates convenient access to well-characterized starter cultures for small scale and commercial Dahi production without dependency on external culture suppliers, a particularly valuable attribute for rural and smallholder dairy producers in India.

Dahi Preparation and Fermentation Studies Dahi Preparation Protocol

Prior to Dahi preparation, the six selected Lactobacillus isolates (NLK, PWA, SNP, PNT, KD, and KA) were inoculated individually into sterile skim milk test tubes using a sterile inoculating loop under aseptic conditions. Each culture was incubated at 37 °C for 24 hours to allow fermentation and acid development in the skim milk. The fermented skim milk culture (inoculum) was then added at the rate of 2% (v/v) to freshly pasteurized milk (500 mL) containing 14% total solids (TS), which was held at 45 °C prior to inoculation. Following inoculation, the milk was mixed gently by swirling and transferred to sterile glass containers. The inoculated milk was then incubated at 37 °C under static (non-agitated) conditions. The fermentation progress was monitored at regular intervals (2-hour intervals) by visual observation of curd formation, whey separation, and pH decline using a calibrated pH meter. Fermentation was considered complete when the curd was fully formed, visually distinct from the whey, and the pH had declined to approximately 4.5-4.8, indicating successful acid development.

Fermentation Duration and Completion Time

The six isolates exhibited variable fermentation kinetics. Isolate NLK completed fermentation in approximately 3.5-4.0 hours, while PWA and SNP completed fermentation in 6.0 hours. Isolates PNT, KD, and KA required 4.5-5.5 hours for complete curd formation and pH stabilization at 37 °C. The observed variation in fermentation time reflects differences in the acidification capacity and metabolic rate among the indigenous Lactobacillus strains. Many Lactobacillus strains exhibit strong lactose catabolism and acidification capacity, enabling rapid pH reduction and improved microbial safety and shelf stability in dairy products (3). The faster-fermenting isolates (NLK, PWA, SNP) demonstrated superior acidification kinetics, while the slower isolates (PNT, KD, KA) produced a firmer, more cohesive curd with better texture retention. Among all six isolates tested, isolate NLK emerged as the best-performing culture, achieving complete fermentation in the shortest time (3.5-4.0 hours) while producing consistently high-quality Dahi with desirable sensory and textural properties. The superior performance of NLK was attributed to its robust acid-producing capacity and efficient lactose fermentation, resulting in rapid pH decline without excessive whey separation or graininess typical of overly rapid fermentation. According to previous findings, during milk fermentation, enzymes released from Lactobacillus cells decompose milk proteins and fats, contributing to desirable changes in texture, flavor, and overall quality of fermented dairy products (Pang et al., 2017). Isolate NLK demonstrated optimal enzyme secretion kinetics, producing a smooth, homogenous curd structure characteristic of premium-quality Dahi.

Sensory Evaluation on 9-Point Hedonic Scale Panel and Evaluation Protocol

Sensory evaluation of Dahi prepared with each of the six Lactobacillus isolates was conducted using a 9-point hedonic scale, where 9 = extremely liked; 8 = very much liked; 7 = moderately liked; 6 = slightly liked; 5 = neither liked nor disliked; 4 = slightly disliked; 3 = moderately disliked; 2 = very much disliked; and 1 = extremely disliked. A trained sensory panel of 10 panelists, comprised of faculty and graduate students familiar with fermented dairy products, evaluated Dahi samples for three key attributes: color, texture (consistency and firmness), and taste (sourness and flavor intensity). The sensory attributes results are presented in Table 2. All Dahi samples were stored at 4 °C overnight and brought to room temperature (approximately 22-25 °C) 30 minutes prior to evaluation to ensure consistent evaluation conditions. Samples were presented in coded glass cups labeled with three-digit random numbers to avoid bias. Panelists were instructed to evaluate each attribute independently and assign a hedonic score based on their perception of acceptability. Distilled water was provided for mouth rinsing between samples. Each panelist evaluated all six samples in a randomized order to minimize sequence bias.

Discussion of Sensory Results

Isolate NLK received the highest mean hedonic scores across all sensory attributes: color (8.2 ± 0.4), texture (8.4 ± 0.5), and taste (8.1 ± 0.6), with an overall acceptance score of 8.2 ± 0.1, corresponding to "very much liked" on the hedonic scale. The superior sensory performance of NLK reflects its optimal fermentation kinetics, which produced a uniformly pale-yellow color characteristic of premium Dahi, a firm yet creamy texture with minimal whey separation, and a mildly sour taste with pleasant lactic flavor. The rapid and efficient acidification by isolate NLK resulted in a smooth, homogenous curd without the graininess or bitterness sometimes associated with overly slow or inefficient fermentation [5] . PWA and SNP performed well, with overall acceptance scores of 7.7 ± 0.4 and 7.5 ± 0.6 respectively, corresponding to "moderately to very much liked." These isolates produced acceptable-quality Dahi with good sensory attributes, though slightly less preferred than NLK. The remaining isolates (PNT, KD, and KA) received moderately acceptable scores (7.1, 6.9, and 6.6 respectively), indicating that while their Dahi products were acceptable, they exhibited some sensory deficiencies compared to NLK, including slightly excessive sourness, weaker texture, or off-flavors. The high scores for isolate NLK demonstrate that indigenous Lactobacillus strains, when properly selected based on fermentation kinetics and metabolic profiles, can produce fermented dairy products of exceptional quality and consumer acceptability. Many Lactobacillus strains possess probiotic characteristics and can contribute food quality and safety by offering multiple organoleptic, nutritional, technological, or health advantages [10] . The success of isolate NLK validates the bioprospecting approach employed in this study and establishes this strain as an excellent candidate for commercial Dahi production and for development as a functional starter culture combining superior fermentation performance with putative probiotic functionality. 7.4 ± 0.2 b 7.5 ± 0.6 ab PNT 7.2 ± 0.4 a 7.1 ± 0.4 ab 7.0 ± 0.4 a 7.1 ± 0.5 ab KD 6.9 ± 0.7 a 6.8 ± 0.5 a 6.9 ± 0.5 a 6.9 ± 0.3 a KA 6.5 ± 0.5 a 6.6 ± 0.2 a 6.7 ± 0.3 a 6.6 ± 0.2 a

Conclusion

This study successfully isolated and characterized 22 indigenous Lactobacillus isolates from Dahi and raw milk samples collected from Haryana, India. The results demonstrate that indigenous Lactobacillus populations in regional dairy sources possess inherent stress tolerance, efficient lactose fermentation, and technological superiority suitable for controlled dairy fermentation. Six isolates demonstrating superior fermentation characteristics were evaluated for Dahi production. Isolate NLK emerged as the best-performing strain, completing fermentation in 6.0 hours at 37 °C and producing Dahi with exceptional sensory quality, achieving the highest hedonic scores for color (8.2 ± 0.4), texture (8.4 ± 0.5), and taste (8.1 ± 0.6). The rapid acidification capacity and optimal fermentation kinetics of NLK, combined with superior consumer acceptability, establish this isolate as a promising candidate for commercial Dahi production. This study contributes to sustainable dairy production systems in India by establishing indigenous starter cultures as viable alternatives to commercial cultures, thereby enhancing food security and livelihoods of rural dairy producers.

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