1. Introduction
Lactic acid bacteria are generally considered safe and have been used in food preservation for several centuries. In the fermented dairy and food industries, LAB make a significant contribution and have an economic impact. There are various studies that have focused on the health benefits of LAB in the modern fermented food processing sector [
1]. The predominant and most commonly found lactic acid bacterial genera are “
Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Vagococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella 1” [
2]. LAB are used to ferment sugar into lactic acid as the major end-product, which further leads to the enhancement of the sensorial, textural, nutritional, and functional attributes of fermented food products [
3,
4]. Lactobacilli are a major and diverse group among all LAB. The main characteristics of the group are that they are Gram-positive, do not form endospores, are catalase-negative, and non-pathogenic, the bacteria belonging to the GRAS category of organisms [
5]. Lactic acid bacteria have reportedly been isolated from a variety of sources, such as raw biotic matrices (grains, crops, plant surfaces, and silages), fermented food products (fermented milk, cereals, fruits, and vegetables), and gastrointestinal and vaginal tracts [
6,
7,
8,
9]. The major application of LAB is in food fermentation to further reduce food spoilage and inhibit pathogenic microbes by virtue of their antimicrobial activities, such as bacteriocin production. Furthermore, their immunostimulatory function and benefits as live probiotic microbes have led to an increasing demand for new indigenous strains with wider benefits [
10,
11,
12]. Each fermented product species provides a distinct environment in terms of competing microorganisms, natural vegetative antagonists, as well as substrate accessibility, type, and concentration of probiotics under various physical factors. Despite these similarities, there are significant differences in substrates and products, as well as the types of microbes used in the production of fermented foods and beverages around the world. Therefore, a number of studies have been conducted to isolate and characterize LAB from diverse and unique natural food matrices [
6].
LAB are used as probiotics due to their various beneficial health effects, i.e., suppression of cancer [
13], antidiabetic activity [
14], allergy prevention [
15], cholesterol-lowering effects [
16], protection from pathogens [
17], immunomodulation [
18,
19], risk-reduction of diseases [
12], and improvement of microflora of the gastrointestinal tract [
20]. Currently, most of the LAB strains used as probiotics belong to the
Lactobacillus and
Bifidobacterium genera [
21] and are used as biocontrol agents due to their mycotoxin-reducing abilities and enhancement of bioavailability [
3]. Probiotic microbes are selected on the basis of various criteria, such as technological attributes (cell growth, stability, and viability in raw food substrates), their effects on the sensorial attributes of products, gastrointestinal tract survivability, and functionality (cell autoaggregation, cell-surface hydrophobicity, bacteriocin production, immunomodulation, antimicrobial activity, and safety) [
22]. However, in vivo studies and clinical trials are required to confirm such properties.
Several fermented foods and beverages are manufactured throughout the world using cereals, with area-specific local variations in terms of composition and processes of manufacturing [
23,
24,
25,
26]. Although India has a wide variety of fermented foods, indigenous fermented product preparation and consumption practices are mostly limited to particular locations and specific communities. Traditional fermented food preparation is one of the world’s oldest biotechnological processes, in which microorganisms play a crucial role in sensory enhancement, enrichment, health-promoting properties, and food preservation [
3,
27,
28].
Raabadi or Rabadi is a popular traditionally fermented cereal-based beverage consumed daily in northwestern India, mainly in rural parts of Haryana, Rajasthan, and Punjab [
29,
30]. It is a very important staple food and provides nutrition and energy to millions of people with low and average incomes in India. A Raabadi-like milk-cereal-based fermented product, “Makka Ki Raab,” is traditionally prepared in Southern Rajasthan, India, which is a very popular drink for people of the region, especially tribals. It is prepared by mixing and fermenting maize flours with “khatti chhaschh” or buttermilk, followed by boiling with continuous stirring for 20–30 min and adding salt as per need. Then, the prepared product is cooled and consumed directly or after mixing with buttermilk as a breakfast beverage. Olia is a curd-rice-like product prepared occasionally in households using cooked rice and Dahi. Rice cooked the previous night is mixed with dahi and khand (raw sugar) in the morning and consumed as a breakfast meal. A Jalebi is a sweet spiral dish widely consumed in the region, prepared using cereal flour, gram flour, and sugar syrup. Kadhi is another cereal-based spiced curd sauce that is prepared using gram flour, buttermilk, and spices. It can be consumed with steamed rice or roti, or pulao.
The probiotic attributes of LAB are strain-specific, and strain characteristics vary from one product to another [
31]. Cereal-based fermented products are recognized as a good source of probiotic strains of LAB. These products contain high counts of LAB, which may be a potential source for exploring beneficial probiotic strains. Although various researchers have isolated, screened, and characterized lactic acid bacterial probiotic strains from cereal-based fermented products in different parts of the world [
28,
32,
33,
34,
35,
36,
37], none of these researchers has studied the cereal-based fermented food products of the Udaipur region (tribal-population-dominant part of Southern Rajasthan, India). Therefore, our investigation aimed to describe, isolate, and evaluate the potential probiotic lactic acid bacterial strains from the cereal-based fermented foods of the studied area.
4. Discussion
The acid tolerances (pH 2.0 or 3.0) of the candidate probiotic LAB strains were evaluated to determine their abilities to survive in the acidic conditions of the stomach [
28,
32]. Ahire et al. 2021 reported that
Lactobacillus plantarum UBLP40 had 73% cell viability at a low pH (2.0) when incubated for 3 h [
32].
Weisella confusa strain GCC_19R1,
Lactobacillus plantarum RPY1,
L. helveticus K14,
Lactobacillus plantarum KJ722784,
Lb. plantarum DM5,
Lb. plantarum LD1,
Lb. plantarum K90, and
Lb. fermantum K75 strains, isolated from different traditionally fermented cereal-based products, also showed good cell viability at low pHs [
28,
33,
52,
53,
54,
55]. Humans have a digestive fluid known as bile which enables the emulsification and solubilization of lipids and lipid-soluble vitamins in the body for proper digestibility [
28,
56]. A higher bile concentration is injurious to the microflora and cell membranes of the host.
Lactobacillus plantarum UBLP40, isolated from traditional Indian fermented products, was exposed to bile (0.3%
w/
v concentration) and cell viability was checked [
32]. Ref. [
57] demonstrated that
Lactobacillus plantarum strain growth decreased with increasing concentrations of bile, which might be due to the detrimental effects of bile. Furthermore, bile tolerance of potential probiotic strains is considered critical for bacterial survival in the gastrointestinal (GI) tract. In this study, at low pHs (2.0 and 3.0), decreasing trends were observed for viability counts with increased exposure times for all isolates. The survivability of isolates was also found to decrease with bile salt concentration (0.3%) up to 3 h exposure. However, the isolates KMUDR7 and KMUDR1 showed minimum decreases in cell counts during the studied period (0 to 3 h). The resistance of many lactic acid bacterial cells might be due to their ability to develop mechanisms to survive in acidic conditions, including stimulation of H+-ATPases, alteration of cell envelopes, ingestion of protons inside cells, and the formation of alkalis [
58]. A typical potential probiotic LAB isolate needs to exert antimicrobial activity against several pathogenic microbes by producing antimicrobial compounds, such as bacteriocin, organic acids (lactic acid, acetic acid, propionic acid), H
2O
2, diacetyl, and surfactants [
50,
51]. Some of the bacteria produce biologically active proteinaceous substances with antimicrobial attributes that are known as bacteriocins. The majority of bacteriocins are effective against Gram-positive bacteria [
59], but some of them also exhibit antimicrobial activity against other spoilage-causing microflora, as well as pathogens [
60]. Recently, [
32] reported the inhibitory potential of
Lactobacillus plantarum UBLP40 against various food pathogens, viz.,
Micrococcus luteus,
Staphylococcus aureus subsp.
aureus,
Pseudomonas aeruginosa, and
E. coli. Some other recent reports are also available regarding the antimicrobial potentials of isolated LAB strains from products of Indian origin [
28,
33,
54,
55,
61]. Some strains, such as
Lactobacillus plantarum (BBC32A, BBC33, BIF43) [
40],
L. helveticus K14 [
54], and
Lactobacillus plantarum KJ722784 [
62], from other cereal-based products have also shown good colonization properties in in vivo tests. Antimicrobial activity is essential for the prevention of pathogens by competitive expulsion in the gut. KMUDR7 showed the highest antifungal activity among all the tested LAB isolates against fungi. Potential probiotic isolates bind to the GI tract and avoid the colonization of fungal species by competitive expulsion and by producing antifungal metabolites [
63]. It is important for a probiotic strain used as a starter culture to provide beneficial effects to the host and extend the shelf life of food [
64]. Recently, various LAB strains isolated from different food matrices were observed with antifungal activities against mycotoxigenic fungi, and efficient strains were identified as
Lb. pentosus and
Lb. sanfranciscensis [
65]. Another study also reported the isolation of the efficient antifungal isolate MYSN 106 from Neera samples [
38].
Further, phenol tolerance at a 0.4% concentration is considered an important attribute for a potential probiotic strain. Phenol is produced in the gastrointestinal tract as a result of deamination of some amino acids and is considered a toxic metabolite for microbial growth. In this study, KMUDR1, -7, and -17 showed phenolic tolerance; however, other strains showed decreasing trends after 24 h exposure to phenol. Previously, other researchers also reported phenol tolerance at 0.4% for the
Lb. plantarum RYPR1 and RYPR9 [
33] and variable tolerance for different LAB isolates [
38,
66]. Lysozyme tolerance in vitro is determined to assess the survivability of strains under stressful conditions of lysozyme exposure, as lysozyme is present in saliva. Selected lactobacilli strains showed high tolerance to lysozyme even after 3 h exposure to lysozyme, with survivabilities of more than 69% [
46]. Other researchers also reported varying degrees of resistance for LAB isolated from fermented foods, with maximum survival values of up to 96.69% [
33,
62]. In our study, KMUDR7 was observed to have the maximum survivability (96.75 ± 1.96%), whereas others showed >53% survivabilities after 60 min exposure, which is in agreement with other studies [
33,
62].
The adhesion properties of bacteria with respect to solvents are used to estimate their abilities to colonize on the gastrointestinal wall. According to [
32], a strain (
Lactobacillus plantarum UBLP40) isolated from fermented foods showed good adhesion to xylene (higher cell-surface hydrophobicity), which was an indication of its good adhesion ability for colonization in the intestine. The adhesion ability of the potential strain to xylene at 30 min was higher in comparison to four
Lb. plantarum strains [
40]. In the current study, KMUDR7 showed the highest cell-surface hydrophobicity (CSH), while the other isolates were observed to have CSHs >55%. Similar trends were also reported for isolated strains, such as
Lb. plantarum RPY1 [
33],
Lb. helveticus K14 [
54], and
Lb. plantarum KJ722784 [
62], from different traditionally fermented cereal-based food products. A potential probiotic strain should have the ability to colonize in the GIT and attach to the intestinal epithelium [
28]. According to [
28], the candidate probiotic strains had varied autoaggregation activities ranging from 16 to 50%. This property helps to prevent the colonization and adhesion of pathogenic microbes. Recently, [
28] evaluated the cell autoaggregation properties of the
Weisella confusa strain GCC_19R1 isolated from sour rice, and the activity was observed to be 38.7%. In the present study, variable degrees of cell autoaggregation (54.89 to 82.47%) were observed for all the tested LAB isolates. The results were in agreement with those for other strains, such as
Lactobacillus plantarum UBLP40 isolated from idli batter [
32] and
Lb. plantarum RPY1 isolated from Raabadi [
33], and strains such as
Lactobacillus plantarum (BBC32A, BBC33, BIF43) [
40],
Lb. helveticus K14 [
54], and
Lb. plantarum KJ722784 [
62] isolated from other cereal-based products also showed good colonization properties in in vivo tests.
The simulated gastric- and pancreatic-digestion experiments were performed to check the survival of LAB isolates in the adverse environment of the gastrointestinal tract. All the tested LAB isolates in the present study showed more than 45% survival, which is in accordance with results for previously reported strains of
Lactobacilli of cereal-based food origin [
32,
33,
40,
62]. Various reports support the claim that
Lactobacilli isolated from cereal-based foods are less resistant to pancreatic juice than gastric juice [
32]. The isolate KMUDR7 showed good survivability (85.54 ± 1.01%) and can be considered a potential probiotic candidate. Using the ABTS method, the relative abilities of LAB isolates to scavenge ABTS were observed to check their antioxidative potentials. The isolates KMUDR1, -7, and -9 showed more than 60% inhibition of ABTS radicals, which indicates their ability to produce radical cations for antioxidant suppression. The antioxidative potential of food-borne probiotic bacteria is supported by various mechanisms, such as the formation of antioxidative compounds (i.e., folate, butyrate, and glutathione), stimulation of antioxidative host genes, suppression of genes associated with reactive oxygen species (ROS) formation, and modulation of gut microflora [
67]. The results of the current study are in agreement with results for LAB isolated from Raabadi [
33].
H
2O
2 production by LAB cultures is also considered beneficial, due to its ability to exert antimicrobial activities against a range of spoilage and pathogenic microbes. H
2O
2 cytotoxicity is due to the formation of certain bactericidal reactive oxides (e.g., hydroxyl radicals) by the action of reducing agents and peroxidases [
68]. Ref. [
33] reported that out of six potential isolates, three strains (RYPR1, RYPR9, and RYPC7) were able to produce H
2O
2. In the present study, two isolates, KMUDR1 and -7, were observed to produce H
2O
2.
BSH activity has also recently been identified as a desirable probiotic feature for strain selection, and the production of BSH enzymes by LAB facilitates the survival of bacteria under high bile acid toxicity conditions. The deconjugation of bile salts by LAB is an indication of host–microbe interaction in the gut, which facilitates the functional regulation of cholesterol metabolism [
67]. Recently, [
57] reported that a
Lactobacillus plantarum strain isolated from foods showed the selective ability to deconjugate bile salts, in contrast to earlier research reports that human-origin strains showed BSH activity. Moreover, this activity was strain-dependent, as a varying degree of bile salt deconjugation was observed among the strains. In the present study, all the isolates, except KMUDR17, precipitated the tested bile salts, which is in agreement with earlier studies [
32,
33].
A probiotic should not produce any toxic substances and should improve safety for consumers by inhibiting pathogens and preventing the production of biogenic amines. These strains should not show hemolytic or DNase activities, these being considered important criteria for the non-virulence of strains. Strains should be resistant to different antibiotics and must not have any transferable antibiotic-resistance genes [
28,
69]. However, highly antibiotic-sensitive probiotic strains with non-transferable resistance genes are considered safe and have been observed to have beneficial effects after antibiotic therapy in terms of eliminating gut pathogens [
32,
70]. Several strains of
Lactobacillus sp. were reported to be safe according to the parameters of antibiotic susceptibility and hemolytic activity [
20,
32,
38,
40]. The results of the present study are in accordance with earlier reports for LAB isolates from cereal-based foods [
32,
33,
54]. Hence, all the tested LAB strains are considered safe potential probiotics based on antibiotic susceptibility profiling, hemolytic activity, and biogenic amine production under in vitro conditions.
Amylolytic LAB are very rare in nature; they contain the amyA gene, which facilitates the conversion of starch into lactic acid by producing α-amylase enzymes. The most proficient starch-fermenting LAB are associated with the genera
Lactobacillus and
Lactococcus [
71]. Three strains of
Lactobacillus acidophilus showing high amylolytic activities (A4, L9, and L23) were isolated from pigs and characterized as potential probiotic candidates [
72]. Recently, [
73] reported three strains of
Lactobacillus plantarum with probiotic attributes possessing high amylase activities isolated from Chinese cereal-based fermented foods. In our study, KMUDR1, -7, and -9 showed weak amylolytic activities. However, amylolytic activities are strain-dependent [
74]. The production of exopolysaccharides (EPSs) is an important desirable property for improving the textural attributes of cereal-based fermented foods without any safety concerns [
75]. Several reports have presented and explored exopolysaccharide-producing bacteria and yeasts isolated from cereal-based products [
36,
44]. The EPS production of
Lactobacillus plantarum isolated from “Nigerian traditional fermented cereal gruel ogi” was investigated by Fourier-transform infrared spectroscopy, and the EPS production for the tested strains was found to be in the range of 1.36 g/L to 2.18 g/L [
44]. In a similar study, thirteen LAB strains isolated from Boza were evaluated for EPS production and potential use as adjunct cultures, and most of the strains showed significant production of EPS [
76]. The production of EPS in foods provides various benefits to living beneficial bacteria, such as protection from adverse conditions, toxic molecules, phagocytosis apart from biofilm formation, and cell recognition [
77]. Various structures of EPSs perform different roles in EPS-producing cells, which are still unknown and mostly strain-dependent [
78]. The six selected isolates were grown in skim-milk media to check their suitability as probiotics and viability during fermentation and storage. All isolates showed growth during fermentation up to 24 h but decreasing trends were observed during storage. The cell populations of all six tested cultures were found to be above the recommended limit of 10
6 CFU/mL for exerting beneficial effects on human health during fermentation. The isolates KMUDR1 and -7 showed acceptable concentrations up to 14 days of storage. Previously, some other researchers [
46,
79] also reported the same trend for a potential probiotic strain.
Morphological, phenotypic, and biochemical characterizations were performed for all six potential probiotic stains to identify the genera of the isolates. Researchers widely use these parameters for the identification of LAB from different indigenously fermented foods products, i.e., Neera samples [
38], Raabadi [
33], yoghurt [
80], and sour rice [
28]. Ref. [
21] reported that the LAB isolates E031, T035, and K011 showed growth at 4% and 6.5% NaCl concentrations, whereas E052 did not grow at a 6.5% NaCl concentration. All the six LAB isolates were further confirmed using phylogenetic tree construction and the 16S rDNA sequencing technique. The four different potential probiotic lactobacilli strains (
Lactobacillus plantarum JCM1149,
Lactobacillus paracasei NBRC15889,
Lactobacillus plantarum CIP103151, and
Lactobacillus paracasei subsp.
tolerans NBRC15906) were identified from indigenously fermented foods products of Ethiopia (Teff dough, Ergo, and Kocho) using 16S rDNA sequencing [
21]. According to [
54], a 16S rDNA gene sequence analysis of the LAB isolates from different fermented foods showed that all efficient probiotic isolates belonged to the
Lactobacilli genus. Similarly, some other researchers have also used this technique for the strain-level identification of potential probiotic strains isolated from various food matrices [
28,
38,
46,
80,
81].