Isolation and Identiﬁcation of Autochthonous Lactic Acid Bacteria from Commonly Consumed African Indigenous Leafy Vegetables in Kenya

: African indigenous leafy vegetables (AILVs) are plants that have been part of the food systems in Sub-Saharan Africa (SSA) for a long time and their leaves, young shoots, ﬂowers, fruits and seeds, stems, tubers, and roots are consumed. These vegetables are high in vitamins, minerals, protein, and secondary metabolites that promote health. This study aimed at isolating, characterizing, and identifying dominant lactic acid bacteria (LAB) from naturally fermenting commonly consumed AILV in Kenya. A total of 57 LAB strains were isolated and identiﬁed based on phenotypic and 16S rRNA gene analyses from three AILVs (23 nightshade leaves, 19 cow-peas leaves, and 15 vegetable amaranth). The highest microbial counts were recorded between 48 h and 96 h of fermentation in all AILVs ranging from approximately log 8 to log 9 CFU/mL with an average pH of 3.7. Fermentation of AILVs was dominated by twenty eight Lactobacillus spp. [ Lactiplantibacillus plantarum (22), Limosilactobacillus fermentum (3), Lactiplantibacillus pentosus (2) and Lactiplantibacillus casei (1)], eleven Weissella spp. ( Weissella cibaria (8), W. confusa (2), and W. muntiaci ) six Leuconostoc spp. [ Leuconostoc mesenteroides (3), Leuc. citreum (2) and Leuc. lactis (1)], six Pediococcus pentosaceus , four Enterococcus spp. [ Enterococcus mundtii (2), E. faecalis (1) and E. durans (1)] and, ﬁnally, two Lactococcus garvieae . These bacteria strains are commonly used in food fermentation as starter cultures and as potential probiotics.


Introduction
African indigenous leafy vegetables (AILVs) inhabit Sub-Saharan Africa (SSA) [1][2][3]. Their leaves, young shoots, flowers, fruits and seeds, stems, tubers, and roots are consumable and they have been part of the food systems for generations [1][2][3]. These vegetables contain high levels of vitamins, minerals, protein, and health-promoting secondary metabolites. This could be a valuable source of nutrition in rural areas, where they can help alleviate malnutrition among poor populations [4].

Isolation of Lactic Acid Bacteria from African Indigenous Vegetables
The microbial counts varied across the three African indigenous leafy vegetables (AILVs). The mean lactic acid bacteria (LAB) count from all AILVs was approximately log 3 cfu/mL at 0 h which slightly increased to approximately log 8 cfu/mL for cowpea and amaranth and log 7 for nightshade leaves after 24 h. The highest microbial counts were recorded between 48-96 h in all AILVs ranging from approximately log 8 to log 9 cfu/mL ( Figure 1). The ability of the bacteria to acidify during fermentation is reflected in the pH production. The mean pH during all AILVs fermentation showed a slow decrease over time from pH 6.3 at 0 h to an approximate mean of pH 4.2 after 24 h. The pH decreased slightly further to a mean of 3.8 after 48 h and stayed at this low level up to 168 h, reaching a mean of 3.5 ( Figure 1).

Phenotypic and Molecular Identification of Lactic Acid Bacteria Strains
A total of 57 strains were isolated and identified according to their phenotypic and molecular characteristics from three African indigenous leafy vegetables (23 nightshade leaves, 19 cowpeas leaves, and 15 amaranth leaves). Phenotypically, the isolates were classified as Gram-positive and catalase-negative, and as either rods or cocci-shaped. Most of the isolates were able to grow in 6.5% NaCl and at 45 • C with a few growing at 10 • C (Tables 1 and 2. Out of 57 isolates, 20 produced gas from glucose fermentation; thus, they were classified as heterofermentative and 37 isolates were homofermentative or did not produce gas from glucose fermentation [18]. Out of these 20 heterofermentative isolates, 6 were coccus-shaped, produced gas from glucose metabolism, and thus were considered either heterofermentative cocci belonging to the genera Leuconostoc or Weissella, whereas 14 strains were rod-shaped and produced gas from glucose metabolism and thus were characterized as heterofermentative rods, which belonged to either the genus Lactobacillus or Weissella. Based on the 16S rRNA gene sequencing results, the GenBank databank showed the six heterofermentative strains that were coccus-shaped and produced gas from glucose fermentation that were identified as Leuconostoc mesenteroides (3 strains) with a sequence similarity of 99%, Leuc citreum (2 strains) with a sequence similarity of 99%, and Leuc lactis (1 strain) with a 99% sequence similarity ( Table 1). The 14 strains that were rod-shaped produced gas from glucose metabolism and were identified with 16S rRNA gene sequencing such as Lactobacillus fermentum (now Limosilactobacillus fermentum) (3 strains), with a sequence similarity of 99%, and 11 strains were identified as Weissella with sequence similarities of 98 to 100% [Weissella cibaria (8 strains), W. confusa (2 strains) and W. muntiaci (1 strain)] (Tables 1 and 2).

Phenotypic and Molecular Identification of Lactic Acid Bacteria Strains
A total of 57 strains were isolated and identified according to their phenotypic and molecular characteristics from three African indigenous leafy vegetables (23 nightshade leaves, 19 cowpeas leaves, and 15 amaranth leaves). Phenotypically, the isolates were classified as Gram-positive and catalase-negative, and as either rods or cocci-shaped. Most of the isolates were able to grow in 6.5 % NaCl and at 45 °C with a few growing at 10 °C (Tables 1 and 2. Out of 57 isolates, 20 produced gas from glucose fermentation; thus, they were classified as heterofermentative and 37 isolates were homofermentative or did not produce gas from glucose fermentation [18]. Out of these 20 heterofermentative isolates, 6 were coccus-shaped, produced gas from glucose metabolism, and thus were considered either heterofermentative cocci belonging to the genera Leuconostoc or Weissella, whereas 14 strains were rod-shaped and produced gas from glucose metabolism and thus were characterized as heterofermentative rods, which belonged to either the genus Lactobacillus or Weissella. Based on the 16S rRNA gene sequencing results, the GenBank databank showed the six heterofermentative strains that were coccus-shaped and produced gas from glucose fermentation that were identified as Leuconostoc mesenteroides (3 strains) with a sequence similarity of 99 %, Leuc citreum (2 strains) with a sequence similarity of 99 %, and Leuc lactis (1 strain) with a 99 % sequence similarity ( Table 1). The 14 strains that were rodshaped produced gas from glucose metabolism and were identified with 16S rRNA gene sequencing such as Lactobacillus fermentum (now Limosilactobacillus fermentum) (3 strains), with a sequence similarity of 99 %, and 11 strains were identified as Weissella with sequence similarities of 98 to 100 % [Weissella cibaria (8 strains), W. confusa (2 strains) and W. muntiaci (1 strain)] (Tables 1 and 2).
Out of 37 homofermentative isolates, 22 isolates were Gram-positive, rod-shaped, catalase negative, did not produce gas from glucose metabolism, and thus were consid-  Out of 37 homofermentative isolates, 22 isolates were Gram-positive, rod-shaped, catalase negative, did not produce gas from glucose metabolism, and thus were considered homofermentative rods belonging to the Lactiplantibacillus (Lactobacillus plantarum) group. These strains were identified as Lactiplantibacillus plantarum with a 16S rRNA gene sequence similarity of 97 to 100%. Two isolates were further identified as Lactiplantibacillus pentosus (Lactobacillus pentosus), with a similarity of 97%, whereas 1 isolate was identified as Lactiplantibacillus casei (Lactobacillus casei) with a sequence similarity of 97% (Table 1). Furthermore, out of 37 homofermentative isolates, 12 were Gram-positive, cocci-shaped, catalase negative, and did not produce gas from glucose fermentation. Based on the 16S rRNA gene sequencing results, 6 isolates were identified as Pediococcus pentosaceus with a sequence similarity of 98 to 100%. Additionally, 4 isolates were identified as Enterococcus, among which 2 were Enterococcus mundtii with a 99% sequence similarity, and E. durans and E. faecalis with percentage sequence similarities of 99% (Tables 1 and 2). Finally, 2 were Gram-positive, cocci-shaped, catalase negative, did not produce gas from glucose fermentation, grew in 6.5% NaCl, and did not grow either at 10 • C or 45 • C. The 16S rRNA gene sequencing identified these isolates as Lactococcus garvieae with similarities of 98 to 100% (Table 1). Based on 16S rRNA genes, fermentation of African indigenous leafy vegetables (AILVs) was dominated by the genus Lactobacillus (Lactiplantibacillus and Limosilactobacillus). In cowpea, for example, the genus Lactiplantibacillus (7) dominated followed by Leuconostoc (6), then Weissella (4) and Enterococcus (2), respectively ( Figure 2). Africa nightshade leaves were dominated by the genera Lactiplantibacillus (9) and Limosilactobacillus (3), followed by the genera Pediococcus (6), Weissella (3), and Enterococcus (2), respectively. Vegetable amaranth was dominated by Lactiplantibacillus (9), Weissella (4), and Lactococcus (2), respectively ( Figure 2). The hierarchical profile among the bacterial species and AILVs revealed two major clusters and three distinct clusters for the isolated bacteria and AILVs, respectively ( Figure  3). Amaranth and cowpeas were clustered separately from the African nightshade. All three vegetables contained Lactiplantibacillus plantarum. Pediococcus pentosaceus was isolated from African nightshades, whereas Weissella cibaria was isolated from cowpea and amaranth ( Figure 3). The hierarchical profile among the bacterial species and AILVs revealed two major clusters and three distinct clusters for the isolated bacteria and AILVs, respectively ( Figure 3). Amaranth and cowpeas were clustered separately from the African nightshade. All three vegetables contained Lactiplantibacillus plantarum. Pediococcus pentosaceus was isolated from African nightshades, whereas Weissella cibaria was isolated from cowpea and amaranth ( Figure 3).

Discussion
Fermentation is a biotechnological process with a long history of application. In Africa, there is little information on leafy vegetable fermentation, as opposed to typical fermentations based on animal or plant protein or starchy plant substrates [5]. In natural or spontaneous fermentation, the conditions are set to encourage the growth of the desirable and most adaptable microorganisms, which produce by-products that aid in outgrowing others and dominating the fermentation [19]. Food fermentation offers numerous benefits, several of which are vital for survival and safe nutrition of populations; for example, it enhances food organoleptic properties, aids in food preservation due to the accumulation of organic acids, CO 2 and bacteriocins from fermenting microbes and reduction of food spoilage, and it also improves food nutritional value through the production of vitamins, essential amino acids, proteins, and fatty acids. It also improves the toxicological safety of products by degradation of antinutritive factors [16,20]. In this study, three commonly consumed African indigenous leafy vegetables (AILVs) in Kenya, i.e., amaranth, cowpeas, and African nightshade leaves were spontaneously fermented in a brine solution of 3% salt and 3% sugar. AILVs are rich in micronutrients, are widely accessible, and are affordable; hence, they were selected for this investigation [6].
The results showed that the spontaneous fermentation of three AILVs was dominated by six distinct populations of lactic acid bacteria (LAB) genera: Lactobacillus (Lactiplantibacillus and Limosilactobacillus), Pediococcus, Weissella, Leuconostoc, Enterococcus, and Lactococcus ( Figure 2). Studies show that plant materials harbor different groups of LAB microbiota, such as Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc mesenteroides, Lactobacillus acidophilus, and Lactococcus lactis strains [5,21]. The participation of LAB in the spontaneous fermentation of various plant materials has already been demonstrated by Kostinek et al. [22], who found several LAB involved in the natural fermentation of cassava. Spontaneous fermentation of kimchi, a Korean fermented vegetable is commonly carried out by the genera Lactobacillus and Leuconostoc [23,24]. Sauerkraut is also produced by the spontaneous fermentation of cabbage by LAB [25,26].
Among the genera, Weissella, Lactococcus, Enterococcus, and Leuconostoc were abundant at the early stages of all three AILVs fermentation (0-72 h). Previous studies reported that these groups of LAB initiate the fermentation when the pH is low and later a more acid-tolerant group, i.e., Lactobacillus, takes over the fermentation. These results agree with our findings where the pH went below 4.0 after 48 h because of LAB growth (Figure 1). In kimchi fermentation, it was reported that Leuconostoc and other less acid-tolerant bacteria dominate the early stages of fermentation [27], but were later replaced by species of the genera Lactobacillus and Weissella, which are better adapted to grow at high acidity and low pH conditions [28,29].
The genera Lactococcus (Lactococcus garvieae) and Enterococcus (Enterococcus mundtii/ Enterococcus faecalis) were among the most abundant LAB bacteria present at 0-48 h. These bacteria belong to a homofermentative group that only produced lactic acid from sugar metabolism [30]. They occur in a wide range of habitats, e.g., on the skin and in the milk and feces of animals, from saliva, breast milk, and the vagina of humans, from plants and vegetables, as well as from a variety of fermented foods [31]. Lactococcus garvieae is commonly used in the manufacture of meat, cheese, and other fermented milk products. It is a good producer of bacteriocins and is hence used in the food industry as a biopreservative agent against various foodborne pathogens [32].
As fermentation progresses, the results showed that members of the genera Lactobacillus, Weissella, and Pediococcus predominated at 24-168 h. Among the LAB isolated, Lactiplantibacillus plantarum (formerly L. plantarum) was the most isolated from 0-144 h (Figures 4-6). Lactiplantibacillus plantarum has been previously isolated from different African fermented foods [20,33,34] and it is known to produce plantaricin [35] with acid-tolerant and probiotic properties. Lactiplantibacillus pentosus (formerly L. pentosus) and Lactiplantibacillus casei (formerly L. casei) are phylogenetically clustered with Lactiplantibacillus plantarum, which were isolated from cowpea leaves at 144 h. Lactiplantibacillus pentosus is used as a starter culture in the fermentation of Spanish-style green olives, contributing not only to the organoleptic properties of the final product but also acting as a biopreservative agent of the fermented olives [36]. Lactiplantibacillus casei is frequently isolated from fermented vegetables, milk, dairy products, and the human intestinal tract. It is generally recognized as safe with a long history of use as a probiotic strain and immune system modulator [37].
Limosilactobacillus fermentum (formerly Lactobacillus fermentum) was isolated from nightshade leaves at 24 h and 144 h, and it is obligately heterofermentative rod-shaped, producing CO 2 from sugar metabolism. It occurs in diverse habitats, including the human gut, milk products, fermenting plant material, and animals [38]. This species is considered a good probiotic candidate because of its ability to withstand gastrointestinal conditions [39]. It was reported to have the potential for the prevention of community-acquired infections, modulation of the immune systems, and production of antimicrobial compounds [40]. Strains belonging to the genus Weissella were isolated at 48 to 72 h, and they included W. cibaria, W. confuse, and W. muntiaci. Members of this genus are Gram-positive, coccoid, or have a rod-shaped morphology [41]. They are obligately heterofermentative, producing CO 2 from sugar metabolism [31]. They occur in a wide range of habitats, e.g., on the skin and in the milk and feces of animals, from saliva, breast milk, feces, and the vagina of humans, from plants and vegetables, as well as from a variety of fermented foods [31]. W. cibaria and W. confusa are linked to the formation of the exopolysaccharide (EPS) dextran, which has prospective applications as a replacement for commercial hydrocolloids in bakery products, as well as health benefits as a prebiotic fiber [42]. Furthermore, members of the genus Leuconostoc were frequently isolated from cowpea leaves at 0-96 h, and they included Leuc. mesenteroides, Leuc. lactis, and Leuc. citreum. Members of this genus are Gram-positive, coccoid, or have a rod-shaped morphology and produced CO 2 from sugar fermentation. Leuconostoc species are involved in several food fermentation processes; for example, Leuc. mesenteroides sub sp. mesenteroides are regular aroma-producing starter cultures in the dairy industry and are involved in vegetable and coffee bean fermentations as well as Leuc. mesenteroides and Leuc. citreum are the dominant species in kimchi fermentation [43]. Additionally, Leuc. lactis is a good producer of EPS with potential applications in the food industry [44]. Pediococcus pentosaceus dominated the fermentation of African nightshade leaves between 48 to 144 h. It is an obligate homofermentative, Gram-positive coccus-shaped LAB [45]. It is frequently utilized in the fermentation of fruits, vegetables, dairy products, meat, and silage [46]. It is known to produce pediocins with broad antimicrobial activity against food pathogens [47] and is crucial in the production of various commercial probiotic feeds [48].

Growth and Preparation of Plant Materials
The African indigenous leafy vegetables nightshade (Solanum scarbrum), cowpea (Vigna unguiculata), and amaranth (Amaranthus retroflexus) (Figure 9) were cultivated at Jomo Kenyatta University of Agriculture and Technology (JKUAT) in Kenya in open fields for six to eight weeks (17-28 • C, 40-50% relative humidity). The vegetables were cultivated during the dry seasons of December to April. The used soil was supplemented with composite farmyard manure with application of drip-line irrigation 12 h a day. The leafy vegetables were harvested by hand-picking and delivered to the laboratory for processing. The leaves were washed with tap water and dried with paper towels.

Fermentation of African Indigenous Leafy Vegetables
Fermentation was performed in 5. L stainless steel buckets. For each vegetable, 1 kg of leaves and 3 L of salt and sugar brine solution were used. The solution consisted of a combination of salt and sugar, 3.0% each. Common table salt (Kensalt ltd, Nairobi, Kenya) and retail sugar (Kabras sugar company ltd, Kakamega, Kenya) were purchased at local stores in Kenya. The brine solution was sterilized by autoclaving for 15 min at 121 °C. Weights were used to hold all plant material below the surface of the liquid. Sampling was done in a sterile setting ( Figure 10). The fermentations were done in duplicates at 25 °C.

Fermentation of African Indigenous Leafy Vegetables
Fermentation was performed in 5. L stainless steel buckets. For each vegetable, 1 kg of leaves and 3 L of salt and sugar brine solution were used. The solution consisted of a combination of salt and sugar, 3.0% each. Common table salt (Kensalt ltd, Nairobi, Kenya) and retail sugar (Kabras sugar company ltd, Kakamega, Kenya) were purchased at local stores in Kenya. The brine solution was sterilized by autoclaving for 15 min at 121 °C. Weights were used to hold all plant material below the surface of the liquid. Sampling was done in a sterile setting ( Figure 10). The fermentations were done in duplicates at 25 °C.

Microbiological Testing
The progress of the fermentation was determined by microbial enumeration and pH determination. The buckets were carefully swirled to mix the fermentation brine and materials followed by collecting 7 mL of brine samples for pH determination and 5 mL for microbiological enumeration in sterile test tubes at 0 h, 24 h, 48 h, 72 h, 144 h, and 168 h. The fermentation samples were mixed by vortexing, and then 1 mL of the mixture was transferred to a 9 mL test tube containing sterile quarter-strength ringer's solution to make serial 10-fold dilutions of 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 , 10 −7 , 10 −8 , and 10 −9 .
These preparations were further mixed by vortexing. Then, 100 µL of the aliquots from different dilutions were spread-plated onto MRS agar (De Man, Rogosa, and Sharpe), M641, Himedia (Mumbai, India) and M17 agar (M929 Himedia (Mumbai, India). The plates were incubated aerobically at 30 • C for 24-48 h. The incubated plates were counted daily for up to 6 days to determine the bacterial colony forming unit (CFU) as described by Wafula and Murunga [49]. For further characterization, colonies were selected at random from the highest dilution agar plates. The isolates were then grown aerobically in MRS broth at 30 • C and streaked to ensure purity. The isolates stock cultures were cryopreserved in MRS broth with 20% glycerol at −75 • C. The isolates were divided into groups based on phenotypic characteristics, and their identity was confirmed through 16S rRNA gene sequencing.

Phenotypic Characterization
Presumptive lactic acid bacteria isolated from cowpea, nightshade, and vegetable amaranth leaves were further characterized. Cell morphology was determined using phasecontrast microscopy at 100× magnification (Shimadzu CX41, Tokyo, Japan). Standardized tests including Gram reaction and catalase activity [50,51], gas (CO 2 ) output from glucose in MRS broth, growth at 6.5% NaCl concentration, and growth at 15 • C and 45 • C were also determined [12]. The strains were classified into three groups based on their phenotypic characteristics: obligately heterofermentative rods, facultatively heterofermentative and obligately homofermentative rods, and obligately heterofermentative cocci.

Genotypic Characterization
Genomic DNA was extracted from overnight cell cultures grown in an MRS broth at 30 • C using a BioWorld Bacterial Miniprep Kit (GeneLink International, Inc., Dublin, OH, USA) according to the manufacturer's instructions. DNA was quantified using a NanoDrop spectrophotometer (PCRmax, Staffordshire, UK). Amplification was performed in a Primus 96 advanced Peqlab, thermal cycler (PEQLAB, Erlangen, Germany) in relation to positions 8-27 and 1511-1491 of the corresponding 16S rRNA gene of Escherichia coli, respectively [52]. The 16S rRNA gene amplification was performed using the universal primers 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1492R (5 -GGT TAC CTT GTT ACG ACT T-3 ).
PCR was performed in a 50 µL mixture containing 25 µL OneTaq ® 2X Master Mix (New England BioLabs, Hertfordshire, UK), 1 µL of each primer, 1 µL of DNA template (10 ng), and 22 µL RNase free water. The reaction mixtures were subjected to the PCR conditions as described by Wafula and Murunga [49]. The amplified PCR products were resolved in 1.2% agarose gel stained with ethidium bromide (1 µg/mL) and visualized using a Uvitec Cambridge gel documentation system (Uvitec, Cambridge, UK). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The purified amplicons were Sanger-sequenced at Human Genomics Macrogen Europe (Macrogen Europe B.V, Amsterdam, The Netherlands).

Phylogenetic Analysis
The 16S rRNA gene sequences of the bacterial isolates were viewed for quality checks and edited using the ChromasPro 2.1.8 software package (http://technelysium.com.au/ wp/, accessed on 13 January 2022). They were then compared with available standard sequences of bacteria lineages in the public nucleotide sequence databases in the National Center for Biotechnology Information (NCBI) using nucleotide blast (https://blast.ncbi. nlm.nih.gov/Blast.cgi, accessed on 13 January 2022) to find closely related bacterial 16S rRNA gene sequences. The 16S rRNA gene sequences of the isolates and those of the unknown closely related bacteria strains were aligned using Clustal W software, and phylogenetic trees were constructed using the Kimura 2-parameter model with MEGA (Molecular Evolutionary Genetics analysis) 7.0 software package [53]. The trees' topologies were evaluated using the bootstrap resampling method [54] based on 1000 replicates. The sequence of Escherichia coli (X80725.1) was used as control.

Data Analysis
The microbial enumeration and pH results were expressed as the mean and standard deviation of duplicate experiments using Microsoft excel 2021. The hierarchical clustering of LAB vs fermentation and the vegetable type were analyzed using Anaconda-Python version 3.0.

Conclusions
Based on 16S rRNA genes, fermentation of African indigenous leafy vegetables was dominated by genera Lactobacillus, Weissella, Leuconostoc, Pediococcus, and Lactococcus. This study revealed that fermented AILVs harbor diverse LAB with Lactiplantibacillus plantarum, Weissella cibaria, and Pediococcus pentosaceus as the dominant species. These bacterial strains could be used in a variety of industrial and commercial settings. Hence, further studies are required to evaluate the isolates' functional properties as potential probiotics and starter cultures. Molecular and metagenomic techniques might be used to thoroughly investigate how the LAB functions during fermentation and microbial variation throughout vegetable fermentation.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.