Isolation and molecular characterization of Lactobacillus delbrueckii subsp based on bulgaricus strain 1 from kefir shows probiotic and antimicrobial properties: Linking probiotics to UNSDG (United Nations Sustainable Development Goals) agenda: 2030

Isolamento e caracterização molecular de Lactobacillus delbrueckii subsp com base na cepa 1 de bulgaricus do kefir mostra propriedades probióticas e antimicrobianas: vinculando probióticos à agenda UNSDG (Objetivos de Desenvolvimento Sustentável das Nações Unidas): 2030

Abstract

The global population increase necessitates the dire need for ample food as medicine. Good health and well-being are stressed on probiotic functional foods. The present study characterizes biochemical and molecular identification of potential lactic acid bacteria. The potent antimicrobial properties also affirm Lactobacillus delbrueckii subsp. bulgaricus strain 3286. Biochemical analysis comprises carbohydrate fermentation, tolerance to acids and bile salts, production of bioactive compounds, lecithinase production, gelatinase production, and strain ripening ability. Antibiotic sensitivity to various antibiotics was assessed employing minimum inhibitory concentration (MIC) and E-test. Strain resistance to increased salt concentrations coherently concludes the positive impact of gut microbiome and gut-brain axis health management. The preliminary assessment requires further in vitro, in vivo, and in silico analysis for commercialization, market strategy and utility as functional food supplementation. The study can be rationalized for sustainable development goals regarding SDG 3: good health and well-being. Further, the UNSDG agenda 2030 also ascertains the role of probiotic foods in life longevity and public health management systems.

Keywords:
lactic acid bacteria; probiotics; antimicrobial resistance; Lactobacillus delbrueckii; SDG: good health and well-being

Resumo

O aumento da população global torna urgente a necessidade de alimentos abundantes como remédios. Alimentos funcionais probióticos são enfatizados para boa saúde e bem-estar. O presente estudo caracteriza a identificação bioquímica e molecular de potenciais bactérias lácticas. As potentes propriedades antimicrobianas também confirmam a cepa Lactobacillus delbrueckii subsp. bulgaricus 3286. A análise bioquímica compreende fermentação de carboidratos, tolerância a ácidos e sais biliares, produção de compostos bioativos, produção de lecitinase, produção de gelatinase e capacidade de amadurecimento da cepa. A sensibilidade aos antibióticos foi avaliada empregando concentração inibitória mínima (MIC) e teste E. A resistência da cepa a concentrações aumentadas de sal conclui coerentemente o impacto positivo do microbioma intestinal e do gerenciamento da saúde do eixo cérebro-intestino. A avaliação preliminar requer mais análises in vitro, in vivo e in silico para comercialização, estratégia de mercado e utilidade como suplementação alimentar funcional. O estudo pode ser racionalizado para objetivos de desenvolvimento sustentável em relação ao ODS 3: boa saúde e bem-estar. Além disso, a agenda UNSDG 2030 também verifica o papel dos alimentos probióticos na longevidade da vida e nos sistemas de gestão da saúde pública.

Palavras-chave:
bactérias do ácido láctico; probióticos; resistência antimicrobiana; Lactobacillus delbrueckii; ODS: saúde e bem-estar

1. Introduction

The word “probiotic” refers to live bacteria applied singly or combined and ingested in balanced amounts to promote health homeostasis and prevent disease (Helmink et al., 2019). Several factors, including strain type, gut colonisation, viability, and stability influence probiotic bacteria’s potency. Combinations of two or more probiotics appeared to have positive health benefits (Yu and Li, 2016). Probiotics are further investigated for prebiotics, synbiotics, and postbiotics to develop healthy human living. Although the importance of food as medicine has long been recognised, probiotics' inherent value and many benefits are only now beginning to be recognised. Probiotics have many sources of isolation and types of protective activity, which keep the advantages and stability inside the human host. Vandenplas et al. (2015) demonstrated how each probiotic strain responds to the requirements of its host. Based on how probiotic food items are used to improve human health, they can be divided into four main groups. The categories are defined by the Food and Drug Administration (FDA) as follows: medication, food ingredients, dietary supplements, and medicinal foods (Degnan, 2008; Hoffman et al., 2008; Ram et al., 2024). Fermented foods, foods based on probiotics, and other functional foods have several health benefits, from relieving depression to treating constipation. However, the main focus is on the effects of ageing and illness, as well as the beneficial effects of probiotic bacteria and healthy gut microbiomes. Realising the benefits of probiotics will prevent negative side effects in every situation. Yoo and Kim (2016) reported that the competitive colonisation of probiotic bacteria, like Lactobacillus found in yoghurt, slows down the growth of pathogens that produce toxins and extends the life span of human hosts. Furthermore, it was clear that employing the best probiotics might improve intestinal regulation by maintaining gut microbiota homeostasis (Yu and Li, 2016). The benefits of probiotics have been linked to the health of the gastrointestinal tract. Lactic acid bacteria in fermented foods and beverages, such as sour milk, kefir, yoghurt, sauerkraut, a range of cheeses, salty seafood, beverages, and pickles, are responsible for preserving traditional values (Amara and Shibl, 2015; Markowiak and Śliżewska, 2017; Moulaei et al., 2024; Ali et al., 2023). Shori et al. (2019) have shown that using dietary supplements, as ancient civilizations did, is the least expensive way to recover from common health problems. Probiotic products such as Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, Enterococcus, Bacillus (Gram-positive bacteria), and Saccharomyces (yeast) are made suitable for human consumption (Markowiak and Śliżewska, 2017). However, the new wave of next-generation probiotics offers a variety of potentially advantageous microbes. It's common to highlight the probiotic advantages of fermented milk, yoghurt, cheese, buttermilk, ice cream, and milk powder. Probiotics can also be found in yoghurt and milk from sheep, goats, or cows. Additionally non-dairy goods such as cereals, soy-based products, fortified fruit beverages, and nutrition wafers are easily found in international markets (Kechagia et al., 2013). The human genome is dwarfed by the diversity of microbes in the gut, allowing for a symbiotic relationship with the human intestine. Mutual functions in development, metabolism, immunity, obesity, hypertension, and homeostasis are noteworthy (Sivieri et al., 2013; Nikbakht et al., 2018; Arifin et al., 2023). Probiotics' anti-obesogenic, antidiabetic, antihypertensive, hypocholesterolemic, hepatoprotective, immunomodulatory, antimicrobial, anti-inflammatory, antiproliferative, and antioxidant qualities have all been demonstrated in clinical research. Contributing to the management of several health conditions such as pouchitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, colon cancer, non-alcoholic fatty liver disorders (NAFLD), etc (Aguilar-Toalá et al., 2018). Probiotics have a dosage compensatory effect, and immunomodulation is an essential and effective way to manage disease (Markowiak and Śliżewska, 2017). Strategies for managing probiotic-associated diseases can be roughly classified into five categories: immunomodulation, antagonists, competitive adherence to the epithelium, suppression of toxin production, and health augmentation (Yoo and Kim, 2016). Prospects for cellular and molecular regulation are presented before basal and superficial homeostasis mechanisms are examined. Molecular pathways can alter the gut microbiota, and the effects on dynamic changes and health are significant (McBurney et al., 2019; Plaza-Diaz et al., 2019; Sanders et al., 2019; Zmora et al., 2019; Davoodvandi et al., 2021). Thus, the utility of lactic acid bacteria corroborates the significance of probiotics in augmenting human health.

Over the years, many researches and clinical trials have been conducted on the effects of probiotics in the prevention and treatment of gastrointestinal diseases, but the potential effects of these symbiotic microorganisms in the intestinal microbiota on psychological diseases have rarely been discussed. The studies that have been conducted on the effect of probiotics in this field are still in the beginning and more studies are needed to determine the efficiency, the effective dose, the time required for the effect and the mechanism of action of different strains of probiotics. The lack of sufficient background in our country raised the need for more research to clarify the efficacy and safety of probiotics in the prevention and treatment of depression and anxiety. According to the contradictory results from past researches, in order to investigate the existence of a relationship between the consumption of probiotics and depression, digestive status and obesity and considering the nutritional style and race, it seems that a clinical study to investigate this effect seems to be harmful.

Lactic acid bacteria (LAB) play a critical role in various industrial processes, including food fermentation and the production of bioactive compounds. Delbrueckii species, particularly Lactobacillus delbrueckii, are widely studied owing to their diverse use and potential health benefits. Understanding the biochemical properties of isolated Delbrueckii strains can provide valuable information about their metabolic capabilities and contribute to developing new biotechnological applications. The present research encompasses the isolation and identification of Delbrueckiistrains. Molecular identification was affirmedusingpolymerase chain reaction (PCR) and DNA sequencing. Carbohydrate Fermentation characterizes the fermentation of glucose, fructose, lactose and maltose using the phenolic red broth test or high-performance liquid chromatography (HPLC). Tolerance to acids and bile salts: Survival and growth of Delbrueckii strains under harsh gastrointestinal conditions is essential for their probiotic potential. Their tolerance to acids and bile salts can be assessed by exposing isolated strains to low pH values ​​and concentrations of bile salts and viability. Production of bioactive compounds was restricted for possessing antimicrobial peptides, exopolysaccharides and bacteriocins. Agar well diffusion assays, thin layer chromatography (TLC), or mass spectrometry were used to understand bioactive compound production. Functional foods and nutraceutical properties were confirmed for enhanced health benefits. Molecular characterization depicted the molecular evolution of Lactobacillus delbrueckii, which was identified in the present study.

2. Materials and Methods

2.1. Isolation of lactic acid bacteria from kefir

Isolation of lactic acid bacteria was preceded, as reported by researchers earlier. In brief, 1 ml of kefir was added with 9 ml of distilled water. The cultures were grown in de meat peptone agaremploying the spread plate technique. Pure cultures were obtained after 24-48 hr incubation at 37 °C (Ismail et al., 2018).

2.2. Biochemical characterization for probiotic properties Gelatinase production

Gelatinase production was performed by adding gelatin to the nutrient medium (at the rate of 10-15 g per 100 ml) and poured into test tubes in a column. Sowing of the test culture was done by injection, immersing the loop with the culture deep into the nutrient medium. The seeded culture was then incubated in a thermostat at an optimal temperature for 1-2 days. Together with the test tubes, two test tubes with an uninoculated culture were placed in the thermostat for control. When recording the results, the intensity of growth and the form of liquefaction of the gelatin medium are taken into account. The gelatinase production was judged by the discoloration of a strip of photographic film placed in a test tube with the culture. Strips of photographic film measuring 25-3 mm, illuminated and developed, are lined with pieces of filter paper, placed in a Petri dish and autoclaved at 110 °C (0.5 atm.) for 30 minutes. The bacterial culture was inoculated into test tubes with MPB; a strip of sterile photographic film was inserted under the tube stopper so that its upper part remains non-immersed in the medium. The culture liquefies the gelatin, the submerged portion of the film becomes transparent and black dust of reduced silver falls to the bottom of the tube. Most gelatinase-producing bacteria discolor the strip within 24 hours. The proposed strain should not liquefy gelatin (Chaudhari et al., 2022).

2.3. Lecithinase production

Lecithinase production was assessed using broth with egg yolk: One egg yolk was aseptically added to 400 ml of sterile MPB, stirred well, poured into sterile test tubes of 4-5 ml each and kept in a thermostat at 37 °C for 24 hr to check for sterility. The medium in the test tubes was confirmed not to become cloudy. One loop of one-day-old culture from a solid nutrient medium was added to a test tube with broth and incubated at 37 °C for 24 hr. After the incubation period, whitish turbidity and floating flakes were recorded, indicating the production of lecithinase by the microorganism. The proposed strain should not produce leticinase (Bindu and Lakshmidevi, 2021)

2.4. Strain ripening ability

One-day-old culture of the test strain was inoculated into sterile milk at a rate of 3-5% of the inoculum to the volume of milk. Test tubes with inoculum and sterile milk without culture (control) were placed in a thermostat and incubated at the optimal temperature for 2-3 days. When recording the results, the ability of the culture to ferment milk and form a clot is taken into account, and the acidity is determined titrimetrically. A number of strains of fermented milk cultures may not ferment milk with the formation of curds but produce substances that change the acidity of the nutrient medium. Therefore, acid formation activity, expressed in Turner degrees (DT), should be determined using the method described in the section (Rodrigues et al., 2021).

2.5. Test for acid-forming activity of the strain

Microorganisms belonging to representatives of lactic acid bacteria, including bifidobacteria, produce various acids that change the pH of the environment (nutrient media in vitro or in the intestines in vivo).The culture was washed off with a 0.9% sodium chloride solution and 2.5 ml of the resulting suspension and added to 25 ml of Blaurock medium or sterile skim milk (inoculation is carried out in 2 test tubes with each medium). The contents are thoroughly mixed and incubated for 48-72 hr at a temperature of (38+/- 1)°C. After incubation, acidity was determined in each test tube (2 parallel samples). Each sample in a volume of 10 ml was titrated with a solution of sodium hydroxide at a concentration of 0.1mol/l. The presence of the phenolphthalein indicator (2-3 drops) until a persistent faint pink color appeared was considered the endpoint. The pH was controlled potentiometrically and in the 8.5 +/- 0.1 range. Acidity was expressed in degrees Turner (DT) and calculated using the Formula 1:

$$\text{DT}=\text{A}\ast \text{K}\ast 10$$ (1)

where

A – The number of milliliters of sodium hydroxide solution, at a concentration of 0.1 mol/l, used for titration;

K – Correction to the titer of sodium hydroxide solution at a concentration of 0.1 mol/l;

DT is a conventional value expressed in milliliters of alkali used to titrate 10 ml of the test suspension.

Example: Titration of 10 ml of suspension required 10.6 ml of sodium hydroxide solution at a concentration of 0.1 mol/l, K = 1.03, then in Formula 2:

$$\text{DT}=10.6\ast 1.03\ast 10=109.18\text{DT}$$ (2)

The average value from two tubes (2 parallel samples from each tube) was calculated, provided that the activity indicators of each of them are close (Nainggolan et al., 2024; Megur et al., 2023)

2.6. Strain resistance test to increased salt concentrations

The culture under study was inoculated into the nutrient medium containing 2.4% and 6.5% NaCl (pH 6.8–7.0) (1 loop – 10 ml of medium) and kept in a thermostat at the optimal temperature for 48 hours. The culture's growth or lack of growth is noted visually (after shaking the tube) by the presence or absence of turbidity and monitored selectively using a microscopic specimen (Ren et al., 2014; Beirami et al., 2024).

2.7. Carbohydrate fermentation

Delbrueckii strains are known for fermenting a wide range of carbohydrates. The utilization of various sugars such as glucose, fructose, lactose, and maltose was examined using various biochemical tests, such as the phenolic red broth test and high-performance liquid chromatography (HPLC).This analysis provides valuable information on the metabolic capabilities of Delbrueckii strains and their potential applications in the production of lactic acid-based products (Kuppusamy et al., 2020).

2.8. Tolerance to acids and bile salts

Survival and growth of Delbrueckii strains under harsh gastrointestinal conditions are essential for their probiotic potential. The tolerance to acids and bile salts was assessed by exposing isolated strains to low pH values ​​and concentrations of bile salts and then determining their viability. Strains with high survival capacity that are desirable for probiotic formulations are affirmative (Ayyash et al., 2021).

2.9. Production of bioactive compounds

Delbrueckii strains produce various bioactive compounds that contribute to their health-promoting properties. These include antimicrobial peptides, exopoly saccharides and bacteriocins. Assessing the production of such compounds involves techniques such as agar well diffusion assays, thin layer chromatography (TLC), or mass spectrometry. Understanding the production potential of these bioactive compounds allows the development of functional foods and nutraceuticals with enhanced health benefits (Amiri et al., 2021).

2.10. Determination of the sensitivity of cultures to antibiotics

Antibiotic sensitivity was determined by serial dilution of the antibiotic in a liquid nutrient medium and diffusion by approved regulatory documents (MUK 4.2.1890-04). The minimum inhibitory concentration (MIC) was determined to characterize the sensitivity of bacteria to antibacterial drugs. MIC was the concentration that suppresses the visible growth of the microorganism under study in a broth culture or on a solid medium. This concentration determines the degree of sensitivity of the strain to the antibiotic. Pheno- and genotypic research methods confirmed pure cultures of microorganisms belonging to a specific species are subject to research to assess antibiotic sensitivity. Serial dilution methods are based on the direct determination of the main quantitative indicator characterizing the microbiological activity of antibacterial drugs (ABP) - the value of its minimum inhibitory concentration (MIC).To determine the MIC, specified concentrations of ABP are added to a nutrient medium, which is then inoculated with a culture of the microorganism under study. After incubation, the presence or absence of visible growth was assessed. Diffusion methods for determining sensitivity are based on the diffusion of ABP from the carrier into a solid nutrient medium and suppression of the growth of the test culture in the area where the concentration of ABP exceeds the MIC. Antibiotic sensitivity employing E-test was preceded by employing a narrow strip of polymer (0.5 - 6.0 cm), onto which a gradient of ABP concentrations (from minimum to maximum) was applied. Inhibition of microorganism growth around the strip was observed. The E-test occurs only in the zone where the concentration of ABP diffusing from the carrier is higher than the MIC, and a drop-shaped zone of inhibition is formed. The MIC value was considered to demarcate the boundary of the growth inhibition zone that comes close to the carrier. The manufacturer supplies detailed instructions for determining sensitivity using E-tests with the reagent kit (Sharma et al., 2017).Antimicrobial susceptibility testing was performed using test cultures comprising gram-negative and gram-positive opportunistic microorganisms Salmonella enteritidis NT and Staphylococ cusaureus 16. Isolates isolated from kefir were tested for the presence of antagonistic activity. The agar diffusion method was used to determine the level of antagonism. Bacterial tests were cultured on MPA medium (meat peptone agar) (Yamaguchi et al., 2013).

2.11. Antagonistic assay

Antagonism against S. enteritidis NT was not detected in the studied lactic acid bacteria isolates. In contrast, areas of growth stimulation of S. enteritidis16 around the wells were noted. Moderately expressed antagonism towards both test cultures was tested. Colored Hiss media with carbohydrates (liquid) was used composed of peptone - 10 g sodium chloride - 5 g carbohydrate - 5-10 g and rede indicator - 10 ml or 1 ml of 1.6% solution of bromothymol blue (pH 7.2 ±0.2). To 1000 ml of distilled water, add 10 g of peptone and 5 g of sodium chloride. Peptone and salt are dissolved by heating the water for several min filtered through a paper filter until the medium becomes completely clear. 5-10 g of one of the tested carbohydrates (lactose, glucose, mannitol, cellobiose, etc.) was added, and then Andrede indicator or bromothymol blue was mixed. The prepared medium was poured into 3 ml tubes along with floats with the sealed end up. The medium was sterilized at 112 °C for 20 min. The floats were filled to the top with a nutrient medium during sterilisation. The prepared medium is colorless or has a pink tint - with the Andrede indicator, grass-green - with the bromothymol blue indicator. The nutrient basis of media for determining the fermentation of carbohydrates is peptone water. Meat broth is not suitable for this purpose, as it contains various carbohydrates, which can lead to false results. The colour of the medium changes due to the growth of bacteria, accompanied by the breakdown of carbohydrates with the formation of acidic decomposition products. With Andrede's indicator, it becomes bright pink, and with bromothymol, it turns blue to yellow. The gas formation in the medium is determined by bubbles collecting in the float (Thompson et al., 2022).

2.12. DNA extraction and molecular identification

Genetic analysis can be performed to study the biochemical properties of Delbrueckii strains further. Whole genome sequencing and comparative genomics can provide insight into different isolates' unique genetic features, metabolic pathways, and potential virulence factors. This information helps understand strain-specific characteristics and facilitates the selection of strains with desired traits for specific applications. Genomic DNA was isolated from sediment samples using the DNA MiniprepKit (QIAGEN) according to the manufacturer’s recommendations. 16S RNA conducted the molecular identification of LAB strains, later confirmed by genome sequencing analysis. To ascertain the sequence identity and GenBank accession number, each sequence amplicon underwent BLAST analysis and alignment with the National Centre for Biotechnology Information (NCBI) Sequence comparison database. A phylogenetic tree was created after para-wise alignment using CLUSTAL W and sequences from the NCBI Gene Bank. To identify the LAB isolates, a neighbor-joining tree was presented and then tested using a bootstrap technique with 1,000 replicates using MEGA 11.0 software (Megur et al., 2023).

3. Results and Discussion

3.1. Isolation and molecular characterization of Lactobacillus delbrueckii subsp. bulgaricus strain 3286 from kefir

12 isolates were obtained from kefir and were classified as lactic acid bacteria. Out of the 12 isolates, four isolates were affirmed as lactic acid producers. One isolate 3(10-3), presumably previously classified as lactic acid bacteria corresponds to yeast. Out of the four isolates, better probiotic efficacy and antimicrobial patterns was ascertained Lactobacillus delbrueckii subsp. bulgaricus strain 3286. The degree of homology with the closest strain MT613595.1:24-778 showed closest similarity with 100.0%. Figure 1 depicts the fasat sequence of the probiotic bacterial isolate.

Figure 1
Lactobacillus delbrueckii subsp. Bulgaricus nucleotide sequence obtained by sequencing the 16S rRNA gene.

Figure 2 and 3 depict the genetic characterization by 16SrRNA and phylogenetic lineage and Lactobacillus delbrueckii subsp. bulgaricus strain 3286 belonging the common genera and species. Figure 3 illustrates the phylogentic evolutionary significance. Biochemical and Molecular characterization was attributed to probiotic and antimicrobial properties. The probiotic efficacies were confirmed using a holistic biochemical methodology. The tests included carbohydrate fermentation, tolerance to acids and bile salts, and production of bioactive compounds in TLC. The proposed strain did not produce lecithinase and also did not liquefy gelatin. Strain ripening ability and strain resistance test to increased salt concentrations confirmed the inherent probiotic nature. In a similar study, employing Lactobacillus plantarum Y44 depicted genotypic and phenotypic characters for potent probiotic properties. Genes underlying the survivability in gastric mucosa was corroborated to bile salt hydrolase, F₀F₁-ATPase, Na⁺/H⁺-antiporter, H⁺/Cl⁻ exchange transporter, cyclopropane-fatty acyl-phospholipid synthase, alkaline shock protein NADH system, glutathione system, and thioredoxin system. Probiotic nature was ascertained to the production of surface proteins and exopolysaccharides (Gao et al., 2020). Biochemical analysis for carbohydrate utilization revealed the affirmed probiotic properties. A similar report showed the usage of fructo-oligosaccharides and inulin by lactic acid bacteria could illustrate synbiotic properties (Renye Junior et al., 2021). Hence, the prebiotic, probiotic and synbiotic properties of lactic acid bacteria utilized in the present study can have a holistic assessment for antidiabetic potentials complicated with related disorders like linked cancers. As our results show the antbiotic resistance pattern, the study could have parallel and distint altered applications. Screening methodologies in demarcating probiotic nature and subsequent community use relies on safety, efficacy and effectiveness (Byakika et al., 2019). Our results demonstrate the preliminary properties in probiotic effects of the lactic acid bacteria isolated from kefir. Moreover, probiotic bacteria have been catalogued against almost all the disorders and diseases, worldwide and research in underway through other underexploited non-treatable conditions, too (Feng and Wang, 2020; Hussain et al., 2023; Vijayaram et al., 2024). Thus, a varietal and variant datasets are available for probiotic property evaluation.

Figure 2
Phylogenetic tree constructed by comparing the 16S rRNA gene of the studied sample with the sequences of reference strains located in the NCBI International Database (NCBI, 2024).

Figure 3
Effect of isolated microorganisms on the growth of bacterial test cultures.

3.2. Antimicrobial susceptibility testing and anibiotc resistance

The most commonly used test cultures were the gram-negative and gram-positive opportunistic microorganisms Salmonella enteritidis NT and Staphylococcus aureus 16. The diameter of the growth inhibition zones of the test cultures was measured (Table 1).

Table 1
Antibacterial activity of lactic acid bacteria.

S.No	Strain	Zone of inhibition (mm)
1	3(10-3) (yeast)	11.5±0.5 13±0
2	4(10-4)	12±1
3	8(10-3)	11.5±1.3
4	11(10-3)	13±1
5	12(10-3)	11.5±0.5

Zones of inhibition of bacterial test growth by selected lactic acid bacteria are shown in Figure 3. Isolated lactic acid bacteria showed weak antagonism against Staphylococcus aureus16.

Isolates 4(10-4) and 11(10-3) were characterized by slightly more pronounced antagonism against Staphylococcus aureus16 compared to other isolates. Maximum zones of growth inhibition 13 mm – 14 mm. However, the zones were not clear and completely transparent, which indicates that the growth of the specified test culture was not completely suppressed.

Antagonism against Salmonella enteritidis NT was not detected in the studied isolates of lactic acid bacteria. In contrast, areas of growth stimulation of S. enteritidis 16 around the wells were noted. Moderately expressed antagonism towards both test cultures was detected in yeast isolates 3(10-3) and 11(10-3).

Susceptibility to nearly seven antibiotcs employed in the study revealed positive benefits devoid of antibiotic resistance and amino acid decarboxylase genes (Gao et al., 2020). The present assessment showed susceptibility of majority strains to 6 antibiotics out of the used 14 antibiotics belonging to class I to VIII. However, further phenotypic, genetic basis and physiological characterization needs to be revisited for confirmatory future applications. Nevertheless, the intriguing patterns coerce to the fact that 8 antibiotics, out of 14 depict antibiotic resistance patterns.

L. delbrueckii subsp. Similarly showed the highest resistance against vancomycin (38.8%), ciprofloxacin (33.3%), and penicillin (27.8%). These results are indicative that antibiotic resistance and transfer of the same needs to be revisited for effective probiotic efficiency (Tavşanli et al., 2021).A study for assessing Italian and Bulgarican strains of L. bulgaricus revealed antibiotic resistance against 10 – 13 antibiotics, out of 24 tested. Moreover, variable patterns of nisin resistance was observed (Tumbarski et al., 2021). Lactobacillus delbrueckii subsp. Lactis depicted effective microbiological cut-offs against ampicillin, gentamycin, erythromycin, and clindamycin (see Table 2). Thus, phenotyping testing of probiotic strains are nowadays emphasized for screening of horizontal transfer of antibiotic resistance genes (Shani et al., 2021). Fermented foods in China was evident for effective screening of antibiotic resistance genes employing pulse field gel electrophoresis. The study illustrated resistance to streptomycin (83.7%) and gentamycin (83.7%) (Yang and Yu, 2019). However, profiling for antibiotic resistance genes by polymerase chain reaction in China catalogued rpoB, erm(B), aadA, bla, cat and vanX as potent genes. Nevertheless, there was no horizontal transfer between bacteria as evident by filter mating assay (Guo et al., 2019). Copious reports have stressed the antimicrobial resistance genes (AMR) and associated horizontal gene transfer conferring probiotic resistance to antibiotics (Moghimi et al., 2023; Rozman et al., 2020; Colautti et al., 2022). Nonetheless, advanced metabolomicsin probiotic efficacy affirmation and similar multiomics approaches are derivative for further commercialization prospects (Mo et al., 2022). Thus, the preliminary study will be preceded for intricate multidisciplinary research in future for confirming the market strategy for development of indigenous probiotics.

Table 2
Sensitivity of lactic acid cultures to antibiotics.

No.	Antibiotics	Diameter of growth inhibition zone, mm
		Class antib.	Conc. µg/disc	1	2	3	4	5	6	7	8	9	10	eleven	12
1	Erythromycin	I	15	19	-	23	24	23	25	20	30	28	26	19	20
2	Streptomycin	II	25	16	-	15	-	11	-	15	-	-	0	-	-
3	Gentamicin		30	16	-	19	20	18	23	15	29	20	18	14	14
4	Amikacin		30	15	12	15	17	16	12	15	12	14	20	8	11
5	Kanamycin		30	0	-	12	-	10	-	11	-	-	eleven	-	-
6	Cefotaxim	III	thirty	10	-	35	-	27	-	10	-	8	8	-	-
7	Cefoperazone		75	12	-	15	10	22	12	15	-	-	14	10	8
8	Cefazolin		30	0	10	20	8	0	10	0	-	10	0	12	8
9	Penicillin- G	IV	10 U	0	11	0	-	0	-	0	-	-	11	-	-
10	Ampicillin		10	19	-	20	-	23	-	26	-	-	9	-	-
11	Rifampicin	V	5	18	26	24	30	24	24	26	25	30	25	30	30
12	Ciprofloxacin	YOU	5	12	-	20	-	14	-	0	-	-	9	-	8
13	Chloramphenicol	VII	thirty	32	22	32	21	27	-	24	19	20	23	24	thirty
14	Tetracycline	VIII	thirty	thirty	-	24	-	27	17	12	22	-	21	-	22

3.3. Linking probiotics to UNSDG (United Nations Sustainable Development Goals) agenda: 2030

Fermented foods in the successful accomplishment of the 17 SDGs enlisted in the UNSDG Agenda has been critically addressed since 2011. Earlier, the concept of probiotics was alienated for attainment of millennium development goals (Irvine and Hekmat, 2011; Franz et al., 2014). Recently, accomplishment of SDGs was also given adequate importance pertaining the UNSDG agenda: 2030 (Tamang et al., 2020). Hence the present study foresees the indirect and indirect effects of probiotics in an interdisciplinary approach. Amongst, the SDGs, 7 goals are linked to probiotics involvement. The respective goals are depicted in Figure 4.

Figure 4
SDGs associated with probiotics meeting UNSDG agenda: 2030.

4. Discussion

SDG 3: Good health and well being renounces the importance of escalated food supply for ever increasing population. Functional foods usage and advocacy for improving supply chain management from developed and developing countries will emphasize accomplishment of SDG 1 and 2. Enhancement of diary fermented probiotics and stress on farming and animal husbandry practices will ensure the SDGs 9,12 and 13. Development of medifoods, diabetic foods and probiotic foods as medicine can have prominent implications in affirming the UNSDG agenda by 2030.

The Dyshlyuk et al. (2022) showed that the most important benefit of probiotics is gut health. If the amount of harmful bacteria in the body increases, it can affect the absorption of nutrients in the food consumed. Therefore, the more useful bacteria there are, the intestines will perform their activity as it should. This may be due to the ability of probiotics to activate specific genes in intestinal cells. Probiotics can reduce abdominal pain, bloating and other digestive symptoms.

Another research by Kumar et al. (2024) showed the beneficial benefit of probiotics is their ability to enhance immune system function. Probiotics have been shown to protect healthy people against cold and flu viruses. According to the results of a study, the use of two types of probiotics can increase the function of the immune system in adults by about 50%. Another study showed that people who used probiotic supplements for 30 days had an increase in T cell production in the face of influenza virus and adenovirus.

Probiotics may be even more active for fitness goals (Khalil et al., 2022). Research () shows that some probiotic strains may even help burn fat. When obese people lose enough weight, their gut microbial flora changes accordingly. This research shows that the gut microbes in obese people allow them to extract more nutrients from food so that more nutrients and calories are absorbed and a lower percentage is excreted in the stool compared to lean people.

Demirok et al. (2023) reported that women who used 2 probiotic strains during pregnancy did not gain much weight in the first 6 months after delivery. Probiotics are also effective in protein absorption. A study showed that using a specific probiotic along with a serving of protein causes more protein absorption. In particular, the researchers concluded that probiotics increased the absorption of the amino acid leucine up to 23%, isoleucine up to 20%, valine up to 70%, glutamine up to 116%, ornithine up to 100%, tryptophan up to 100% and citrulline up to 128%. They gave. The higher the amount of amino acid taken from the protein, the higher the muscle growth. Taking BCAA, glutamine and citrulline along with probiotics containing Bacillus coagulans can also increase its effects.

Probiotics improve the response to physical and psychological stress, meaning they act much like an adaptogen. This suggests that it may affect the body's neurotransmitters and hormones, which can enhance the brain's mood function. In fact, a study conducted by Irish researchers found that mice receiving certain probiotics had lower cortisol levels and less anxiety and depression behaviors. The study by Yu et al. (2024) showed similar results in humans. And in a recent study on male rats, those who ate a diet rich in probiotic yogurt were more fertile. These results may be due to higher testosterone production in mice receiving probiotic kefir.

5. Conclusion

The present preliminary study aims to identify potent probiotic strains from Kefir. 12 isolates were obtained, out of which 4 strains were affirmative for probiotic properties and gut survival capability. One strain was demarcated as yeast due of cultural characteriistics. 4 lactic acid bacteria were screened for probiotic efficacy and gut survival. Biochemical studies selected a single strain with positive carbohydrate fermentation, tolerance to acids and bile salts, production of bioactive compounds, lecithinase production, gelatinase production, and strain ripening ability. Genetic analysis by 16SrRNA sequencing revealed 100% homology to Lactobacillus delbrueckii subsp. bulgaricus strain 3286. Antimicrobial susceptibility testing and antibiotic resistance by MIC and E-test proved variability patterns against the group I-VIII antibiotics tested. Concrete confirmations for antimicrobial genes in the strain and horizontal gene transfer mechanisms requires further assessments. UNSDG agenda 2030 was taken in to account. Out of the 17 goals enlisted for accomplishment, the probiotics significance was correlated for direct and indirect influences. 7 goals were ascertained for multidisciplinary prominence enabling future research and comprehensive assessment involving multitude arenas, ranging from antimicrobial resistance to metabolomics and multi-omics.

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Publication Dates

• Publication in this collection
  07 Feb 2025
• Date of issue
  2024

History

• Received
  25 May 2024
• Accepted
  23 Aug 2024