Respiration-competent indigenous Lactobacillus from traditional Dahi and plant sources: Functional characterization for dairy industry applications

Introduction

Species within the genus Lactobacillus have a long history of safe consumption and are globally recognized as premier probiotic microorganisms owing to their well-documented health benefits [1] . Beyond their therapeutic value, indigenous Lactobacilli are deeply intertwined with the natural microbial ecology of fermented foods, fresh plant matrices, and traditional dairy ecosystems. Despite their ubiquity, many of these native, wild-type strains possess highly desirable technological and functional traits that remain largely unexploited for commercial starter culture development and industrial bioprocessing. In modern dairy manufacturing, the commercial exploitation of these native strains requires comprehensive evaluation of their industrial robustness. The ultimate commercial viability of an industrial starter culture depends not only on primary acidification kinetics but also on critical processing parameters, including high biomass productivity, long-term storage stability, oxidative stress tolerance, and robust survival during rigorous downstream preservation techniques such as freeze-drying. These requirements have renewed interest in metabolic strategies that can enhance the robustness of lactic acid bacteria before their deployment in industrial systems [2] . Lactobacilli have traditionally been regarded as primarily fermentative organisms, relying heavily on substrate-level phosphorylation and generally lacking the complete respiratory machinery found in classical aerobic bacteria. However, this perspective has changed substantially. Several strains are now known to adopt a respirationInternational Journal of Advanced Biochemistry Research https://www.biochemjournal.com like metabolism when oxygen is available in conjunction with exogenous heme and menaquinone sources, enabling the activation of electron transport-linked functions that enhance growth and physiological robustness. In Lactobacillus plantarum WCFS1, supplementation with both heme and menaquinone increased biomass production and altered end-product formation, demonstrating that respiratory phenotypes can be induced through external cofactor provision. Similar benefits have been documented in Lactobacillus casei, where respiratory cultivation improved survival against oxidative stress and freeze-drying stress, rendering this approach attractive for industrial starter culture production [2,4] . The present study isolated and phenotypically characterized Lactobacilli from local dairy and plant samples, then screened them for growth enhancement under heme-and menaquinone-supplemented conditions. Rather than presenting this dataset as a simple catalog of isolates, the work constitutes a preliminary bioprospecting exercise for identifying starter culture candidates with respiratory potential. This perspective is essential because the central scientific and industrial question is not merely whether an isolate exhibits improved growth in enriched MRS, but whether respiratory conditioning can be leveraged to generate superior starter cultures optimized for dairy manufacturing (Ricciardi et al., 2019; Zotta et al., 2017). Accordingly, the objective of the present study is to characterize this indigenous collection through the lens of starter culture functionality and industrial applicability. Primary emphasis is placed on identifying respirationresponsive native Lactobacillus isolates, documenting phenotypic strain heterogeneity under oxidative and processing stress conditions, and recognizing topperforming candidates. Ultimately, this work demonstrates how these robust, high-performing indigenous strains can be strategically advanced toward industrial applications, including high-density concentrated inoculum production, development of innovative fermented milk products, and design of stress-resilient probiotic adjunct cultures for modern functional dairy systems [2,5] .

Materials and Methods

Media and Reagents

All media used in the present study were prepared with distilled water and sterilized by autoclaving at 121 °C and 15 psi for 15-20 minutes. Media were obtained from Hi Media Laboratories Pvt. Ltd., Mumbai, India. Hemin was used as the heme source for inducing aerobic respiration, while menaquinone (Supelco, Bellefonte, PA, USA) was added as the electron carrier supplement in MRS broth.

Sample Collection

A total of 60 samples comprising 40 dairy samples and 20 vegetable samples were collected from rural and urban areas of Chhattisgarh, India. All samples were collected aseptically in sterile containers and transported to the laboratory for immediate processing. Dairy samples consisted of Dahi (traditional curd), while vegetable samples included cucumber, spinach, brinjal, chilli, tomato, broccoli, cabbage, ridge gourd, bottle gourd, and lady finger.

Isolation and Preliminary Phenotypic Identification

Dahi samples were homogenized in sterile saline prior to serial dilution, while vegetable samples were first enriched in MRS broth and subsequently plated following dilution.

Colonies with typical translucent to whitish appearance were selected from MRS agar plates after incubation at 37 °C for 24-48 h and transferred to broth for purification. Purity was confirmed by microscopy, and isolates displaying characteristics consistent with lactic acid bacteria were retained for further investigation. Phenotypic identification employed a polyphasic approach based on cell morphology, Gram reaction, catalase activity, and carbohydrate utilization patterns. Gram staining revealed that all selected isolates were Gram-positive rods, consistent with the morphology expected for Lactobacillus species.

Catalase activity was tested with 3% hydrogen peroxide to differentiate typical catalase-negative lactic acid bacteria from unrelated contaminants. Carbohydrate fermentation profiles were generated using a commercial Hi-Carbo kit containing twelve immobilized carbohydrates. Based on this polyphasic characterization, 36 isolates were retained as Lactobacillus cultures for further physiological evaluation.

Assessment of Respiration-Competent Lactobacillus Isolates

Respiration-promoting medium was prepared by supplementing MRS broth with 2.5 µg/ml hemin and 1.0 µg/ml menaquinone. Hemin stock was prepared in 0.05 M NaOH, while menaquinone stock was prepared in ethanol and added aseptically following sterilization of the broth. This formulation is consistent with respiration-inducing conditions employed in related literature for selected Lactobacilli.

Functional Screening for Respiratory Response

Freshly activated cultures were inoculated at 2% v/v into unsupplemented MRS broth and into heme-and menaquinone-supplemented MRS (MRS-HM) for comparative growth assessment. Incubation was performed at 37 °C under both static and shaking conditions, and growth was evaluated spectrophotometrically (UV-Vis 119, Systronics Pvt. Ltd. Ahmedabad, India) at OD600 following incubation. OD600 measurement was used as a preliminary screening metric for respiratory responsiveness rather than as a definitive measure of culture performance. Isolates exhibiting higher optical density under supplemented conditions were categorized as respiration-responsive and selected as candidates for future starter culture development based on their demonstrated respiratory competence.

Data Interpretation Strategy

The original dataset emphasized isolate-level differences in OD600 between control and supplemented conditions. In the present analysis, these data are interpreted functionally. A positive growth differential in the presence of hemin and menaquinone is interpreted as evidence of respiratory conditioning potential, whereas negligible or negative responses indicate strain-specific non-responsiveness or hemin sensitivity, as also documented in the literature. The strongest-performing isolates are therefore positioned as candidates for subsequent application studies rather than as standalone endpoints.

Results and Discussion

Isolation of indigenous Lactobacilli from dairy and plant-associated niches

The isolation procedure yielded presumptive lactic acid bacteria from Dahi and plant-associated samples collected from different locations of Chhattisgarh. Following purification and phenotypic characterization, 36 isolates were identified as members of the genus Lactobacillus based on their Gram-positive rod morphology, predominantly catalase-negative reaction, and carbohydrate fermentation profiles characteristic of the genus as presented in Table 1 and Figure 1. The successful recovery of these isolates confirms that traditional fermented dairy products and fresh plant materials constitute valuable natural reservoirs of indigenous Lactobacillus strains with potential technological significance. This observation is consistent with previous reports demonstrating that naturally fermented dairy products harbor diverse populations of Lactobacillus because spontaneous fermentation, nutrient-rich milk composition, and acidic conditions selectively favor the growth of lactic acid bacteria [6,7] . Similarly, fresh vegetables and other plant materials have been recognized as important ecological niches for epiphytic lactobacilli, where these bacteria adapt to fluctuating environmental conditions such as oxygen availability, nutrient limitation, and variable pH [8,9] .

Respiration screening on isolates

All the 36 phenotypically identified isolates were successfully revived and subjected to respiration-promoting growth conditions using MRS broth supplemented with heme and menaquinone. Fourteen isolates exhibited a marked increase in optical density compared with the unsupplemented control, indicating that approximately 39% of the indigenous Lactobacillus collection benefited from respiratory conditioning. The heterogeneous growth responses observed in the present study further emphasize the strain-specific nature of respiratory metabolism. While several isolates exhibited substantial increases in optical density under supplemented conditions, others showed only marginal improvement or even reduced growth relative to the control. Such variability has also been reported by (2), who demonstrated that only selected Lactobacillus strains exhibited significant respiratory enhancement following heme and quinone supplementation, whereas other strains remained unaffected despite belonging to the same genus.

Superior Respiratory Growth Potent Isolate

Among the respiration-responsive isolates evaluated under heme-and menaquinone-supplemented MRS broth, isolate Krb2 exhibited the highest growth response, attaining an OD600 of 1.054 compared with 0.501 in the corresponding unsupplemented control. This marked increase in biomass indicates that Krb2 possesses a highly efficient respiratory metabolism under the tested conditions, enabling improved energy conservation and enhanced cell proliferation during aerobic cultivation. Respiratory metabolism in certain Lactobacillus strains is known to enhance ATP generation through the establishment of a functional electron transport chain in the presence of heme and quinone, thereby promoting higher biomass yields and increased tolerance to oxidative stress [2,4] . The superior performance of Krb2 therefore suggests that this isolate may possess the genetic and physiological machinery required to efficiently utilize respiratory cofactors, making it a promising candidate for industrial biomass production. Increased biomass generation is particularly advantageous for starter culture manufacture, as it can improve inoculum yield, reduce production costs, and enhance the viability of cultures during downstream processing [10] . Besides Krb2, isolates jdpb, dmtr, Surj, and Chil nw also exhibited appreciable increases in optical density under respiration-promoting conditions, indicating that respiratory competence is distributed among several indigenous isolates rather than being restricted to a single strain. Such variability is expected because respiratory capacity in Lactobacillus is a strain-dependent characteristic influenced by differences in respiratory gene clusters, cofactor utilization, and oxidative stress response systems [2,3] . The identification of multiple respiration-responsive isolates is advantageous from a technological perspective because selection of starter cultures should consider several functional traits in addition to biomass production. Although Krb2 demonstrated the highest growth response, other isolates may possess superior characteristics with respect to acidification rate, flavor compound production, stress tolerance, or probiotic functionality. Similar observations have been reported by Ricciardi et al. [5] , who emphasized that the technological suitability of lactic acid bacteria depends on the combined expression of multiple desirable traits rather than a single growth parameter. Therefore, the present findings support the development of a diverse panel of indigenous starter candidates for subsequent technological characterization instead of relying exclusively on a single isolate.

Respiratory Competence Distribution across Multiple Indigenous Isolates

Comparison of cultivation conditions further revealed that respiratory enhancement was generally more pronounced during static incubation than under shaking conditions. Several isolates exhibited greater differences between supplemented and control cultures in static cultures, whereas agitation either reduced the magnitude of the response or eliminated it altogether. This observation suggests that oxygen availability and aeration must be carefully balanced to achieve optimal respiratory metabolism. Moderate oxygen levels supplied under static cultivation may be sufficient to activate the electron transport chain in respiration-competent lactobacilli, whereas excessive oxygen transfer during shaking can increase the generation of reactive oxygen species, thereby imposing oxidative stress that counteracts the beneficial effects of respiratory metabolism [2,4] . Similar findings have been reported by Zotta et al. [3] , who demonstrated that respiratory performance in lactic acid bacteria is strongly influenced by the interaction between oxygen concentration, heme availability, and redox balance. Consequently, the expression of respiratory metabolism is not determined solely by supplementation with heme and menaquinone but also depends on cultivation parameters such as aeration, agitation rate, inoculum density, and medium composition. These findings highlight the importance of process optimization for maximizing biomass production in respiration-competent indigenous Lactobacillus strains and provide a basis for future bioprocess studies aimed at improving starter culture propagation under controlled aerobic conditions.

Conclusion

This study interprets as a functional bioprospecting effort for starter-culture improvement rather than only a descriptive isolation exercise. Among 36 phenotypically confirmed Lactobacillus isolates recovered from Dahi and plant-associated samples, 14 displayed improved growth under heme-and menaquinone-supplemented conditions, demonstrating that respiratory responsiveness is common but clearly strain dependent in indigenous Lactobacilli. Isolate Krb2 showed the strongest response and therefore represents a priority candidate for deeper evaluation as a high-biomass and potentially stress robust culture. Taken together with published evidence that respiratory cultivation can improve biomass yield, oxidative tolerance, and preservation survival in selected Lactobacilli, these findings support respiratory conditioning as a practical platform for developing stronger starter and adjunct cultures from local microbial resources. The study thus lays a foundation for a next generation of work focused on molecular confirmation, process optimization, and dairy performance validation under starter-manufacturing conditions.

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