**1. Introduction**

Lactic acid bacteria (LAB) are a versatile group of microorganisms and with a long history of use in fermented foods. LAB are distributed in diverse environments and are of considerable economic interest due to their "Food Grade Status" [1].

LAB have been extensively used as starter cultures, as probiotics, and in the production of inhibitory compounds. The latter has been considered as a new generation of antibiotics [2].

The growing number of people with lactose intolerance, allergy to cow's milk protein, or high cholesterol, which cannot consume dairy products, is affected by the lack of access to beneficial probiotic bacteria. Thus, fermented fruits or vegetables, which are an alternative source of probiotic bacteria, have received considerable attention from the consumers. The increasing trend for veganism or vegetarianism has promoted an increase in consumer interest for functional foods that contain beneficial bacteria [3].

When evaluating aspects of safety, taxonomy, and potential to produce pathogenic toxins and resistance to antibiotics, LAB have been recognized by the Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) as "generally recognized as safe" (GRAS) [4]. Although resistance genes in bacteria, including Lactobacillus, are located in transposable elements that can be transferred to other species, the US legislation has no guidelines that contemplate the potential development of resistance for microorganisms used in foods [5]. Alternatively, the European Food Safety Authority (EFSA) established guidelines that define the safety standards for microorganisms used "from farm to fork" [6]. The probiotics belonging to the species included in the category "qualified presumption of safety" (QPS) by EFSA show excellent safety records [4]. Undoubtedly, a full safety assessment of newly identified strains begins with proper identification and an in vitro evaluation of the potential risks [7].

Numerous research studies have attempted to design fermented foods based on fruits or vegetables with probiotic bacteria. LAB can display a remarkable degree of phenotypic and genotypic diversity, allowing them to survive in a variety of habitats and stress conditions. Most species in the LAB group are found colonizing the human or animal intestine but also in several fermented foods, vegetables, and fruits [8, 9, 10].

The capacity to adapt to environmental changes depends on the genetic repertoire and the competency to use micronutrients along with the ability to counteract and overcome externally exerted physical-chemical challenges [11]. Despite an immeasurable arsenal of microbes, each plant or fruit harbors a distinctive microbiota that represents a remarkable niche to several LAB species [12]. However, the microbial composition in these environments is fluctuating and depends on intrinsic (physical and nutritional conditions) and extrinsic (environmental and harvesting conditions) parameters of the plant matrix [13]. Depending on the plant or fruit origin, the LAB species differ; thus, the *Leuconostoc, Lactobacillus, Weissella, Enterococcus,* and *Pediococcus* genera are those most frequently identified as epiphytes within the microbiota [14]. Although several species have been identified and characterized, the selection of new strains with valuable biotechnological properties remains a topic of interest [2].

Ecuador is known as a megabiodiverse country, where access to genetic resources for research is still deficient. Studies on the microbial diversity of unexplored niches and environments are of interest as these might lead to the identification of an endless number of species with unique characteristics and properties. It has been claimed that the microbial population present in raw plant material that has been originated from unexplored niches, differs among samples, as the plant matrix might carry a particular microbiota in a specific geographical region and at a specific time-point [15].

Recently, we proposed to investigate the microbiota associated with wild fruits; especially, to select and characterize strains of beneficial lactic acid bacteria [16]. Tropical forest fruits provide essential nutrients, minerals, and antioxidants that keep a healthy body and provide resilience to disease [17]. The physical and

**17**

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity…*

nutritional properties of fruits, together with the environmental and harvesting conditions of the plant matrix, may influence the final microbial community composition [2]. However, we hypothesize that the wild fruits from the Amazon region differ in their native availability of nutrients and physicochemical conditions, especially when compared to domesticated fruits from commercial crops. These substantial differences might influence the range of potential microbiota.

yet, the lack of research makes this natural resource an yet unknown source of benefits to the food industry [18]. The prevention of diseases through appropriate nutrition with such fruits or the active substances extracted from such fruits or the products from the associated bacterial community can save lives and income [19]. The role of LAB isolated from fruits is not clearly understood; nonetheless, as they are naturally present on the surface of fruits, they produce antimicrobial compounds that might be used as a biological agent to control the growth of spoilage bacteria, and without changing the sensory properties of foods [20, 21]. Thus, we expected to find microorganisms with unique characteristics unlike those of the strains found in ordinary fermented materials, such as fermented milk or vegetables. Nonetheless, to use such strains as part of probiotic foods implies the application of numerous functional analyses to determine their susceptibility or resistance to antibiotics, capacity to prevent the growth of harmful bacteria by competitive

Wild tropical fruits are used for nutrition and medicine by the local inhabitants;

The resistance of LAB to antibiotics is a relevant biosafety issue. Some strains with intrinsic antibiotic resistance could be useful to restore the antibiotic resistance of the gut microbiota after antibiotic treatments [22]. Specific antibiotic resistance genes are carried by transposable elements and constitute a reservoir of resistance for potential food or gut pathogens, thus representing a major biosafety issue [22, 23]. In this regard, special attention should be given to the presence of antibiotic resistance determinants and their potential mobility. Lactobacilli are usually sensitive to the cell wall-targeting penicillin and β-lactamase, but more resistant to cephalosporins. Also, some species are susceptible to low concentrations of several inhibitors of protein synthesis, such as chloramphenicol and tetracycline [22]. Some LABs are considered the most promising natural food preservatives, as they secrete antimicrobial substances (i.e. bacteriocins) that, when applied as crude-extracts to food, precipitated peptides that protect the products from deterioration by microbial activity [24]. LAB can also be used in the pharmaceutical industry to produce compounds such as esters, through the reduction of fructose, as

This research aimed to investigate the lactic acid bacteria diversity associated with the microecosystem of wild fruits from the Amazon region of Ecuador. We sought to find unique characteristics, unlike those of the strains found in ordinary fermented materials such as milk or vegetables. As a first step to select the most promising strains with superior functional properties, we present broad insights into this microenvironment. To our knowledge, this is the first study that assessed the diversity and functional properties of LAB in native tropical fruits from the Amazon region of Ecuador.

The source wild fruit samples were collected during the rainy season (April–July,

2014-2015) from the tropical forest of the Sucumbíos Province in the Amazon region of Ecuador (**Figure 1**). Several units of fruits from *Chrysophyllum oliviforme,* 

*DOI: http://dx.doi.org/10.5772/intechopen.94179*

exclusion, and production of organic compounds.

the most abundant sugar present in fruits [25].

**2.1 Sampling, isolation, and purification of lactic acid bacteria**

**2. Materials and methods**

#### *Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity… DOI: http://dx.doi.org/10.5772/intechopen.94179*

nutritional properties of fruits, together with the environmental and harvesting conditions of the plant matrix, may influence the final microbial community composition [2]. However, we hypothesize that the wild fruits from the Amazon region differ in their native availability of nutrients and physicochemical conditions, especially when compared to domesticated fruits from commercial crops. These substantial differences might influence the range of potential microbiota.

Wild tropical fruits are used for nutrition and medicine by the local inhabitants; yet, the lack of research makes this natural resource an yet unknown source of benefits to the food industry [18]. The prevention of diseases through appropriate nutrition with such fruits or the active substances extracted from such fruits or the products from the associated bacterial community can save lives and income [19]. The role of LAB isolated from fruits is not clearly understood; nonetheless, as they are naturally present on the surface of fruits, they produce antimicrobial compounds that might be used as a biological agent to control the growth of spoilage bacteria, and without changing the sensory properties of foods [20, 21]. Thus, we expected to find microorganisms with unique characteristics unlike those of the strains found in ordinary fermented materials, such as fermented milk or vegetables. Nonetheless, to use such strains as part of probiotic foods implies the application of numerous functional analyses to determine their susceptibility or resistance to antibiotics, capacity to prevent the growth of harmful bacteria by competitive exclusion, and production of organic compounds.

The resistance of LAB to antibiotics is a relevant biosafety issue. Some strains with intrinsic antibiotic resistance could be useful to restore the antibiotic resistance of the gut microbiota after antibiotic treatments [22]. Specific antibiotic resistance genes are carried by transposable elements and constitute a reservoir of resistance for potential food or gut pathogens, thus representing a major biosafety issue [22, 23]. In this regard, special attention should be given to the presence of antibiotic resistance determinants and their potential mobility. Lactobacilli are usually sensitive to the cell wall-targeting penicillin and β-lactamase, but more resistant to cephalosporins. Also, some species are susceptible to low concentrations of several inhibitors of protein synthesis, such as chloramphenicol and tetracycline [22]. Some LABs are considered the most promising natural food preservatives, as they secrete antimicrobial substances (i.e. bacteriocins) that, when applied as crude-extracts to food, precipitated peptides that protect the products from deterioration by microbial activity [24]. LAB can also be used in the pharmaceutical industry to produce compounds such as esters, through the reduction of fructose, as the most abundant sugar present in fruits [25].

This research aimed to investigate the lactic acid bacteria diversity associated with the microecosystem of wild fruits from the Amazon region of Ecuador. We sought to find unique characteristics, unlike those of the strains found in ordinary fermented materials such as milk or vegetables. As a first step to select the most promising strains with superior functional properties, we present broad insights into this microenvironment. To our knowledge, this is the first study that assessed the diversity and functional properties of LAB in native tropical fruits from the Amazon region of Ecuador.

### **2. Materials and methods**

#### **2.1 Sampling, isolation, and purification of lactic acid bacteria**

The source wild fruit samples were collected during the rainy season (April–July, 2014-2015) from the tropical forest of the Sucumbíos Province in the Amazon region of Ecuador (**Figure 1**). Several units of fruits from *Chrysophyllum oliviforme,* 

*Ecosystem and Biodiversity of Amazonia*

that contain beneficial bacteria [3].

evaluation of the potential risks [7].

properties remains a topic of interest [2].

specific time-point [15].

antibiotics [2].

LAB have been extensively used as starter cultures, as probiotics, and in the production of inhibitory compounds. The latter has been considered as a new generation of

The growing number of people with lactose intolerance, allergy to cow's milk protein, or high cholesterol, which cannot consume dairy products, is affected by the lack of access to beneficial probiotic bacteria. Thus, fermented fruits or vegetables, which are an alternative source of probiotic bacteria, have received considerable attention from the consumers. The increasing trend for veganism or vegetarianism has promoted an increase in consumer interest for functional foods

When evaluating aspects of safety, taxonomy, and potential to produce pathogenic toxins and resistance to antibiotics, LAB have been recognized by the Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) as "generally recognized as safe" (GRAS) [4]. Although resistance genes in bacteria, including Lactobacillus, are located in transposable elements that can be transferred to other species, the US legislation has no guidelines that contemplate the potential development of resistance for microorganisms used in foods [5]. Alternatively, the European Food Safety Authority (EFSA) established guidelines that define the safety standards for microorganisms used "from farm to fork" [6]. The probiotics belonging to the species included in the category "qualified presumption of safety" (QPS) by EFSA show excellent safety records [4]. Undoubtedly, a full safety assessment of newly identified strains begins with proper identification and an in vitro

Numerous research studies have attempted to design fermented foods based on fruits or vegetables with probiotic bacteria. LAB can display a remarkable degree of phenotypic and genotypic diversity, allowing them to survive in a variety of habitats and stress conditions. Most species in the LAB group are found colonizing the human or animal intestine but also in several fermented foods, vegetables, and fruits [8, 9, 10]. The capacity to adapt to environmental changes depends on the genetic repertoire and the competency to use micronutrients along with the ability to counteract and overcome externally exerted physical-chemical challenges [11]. Despite an immeasurable arsenal of microbes, each plant or fruit harbors a distinctive microbiota that represents a remarkable niche to several LAB species [12]. However, the microbial composition in these environments is fluctuating and depends on intrinsic (physical and nutritional conditions) and extrinsic (environmental and harvesting conditions) parameters of the plant matrix [13]. Depending on the plant or fruit origin, the LAB species differ; thus, the *Leuconostoc, Lactobacillus, Weissella, Enterococcus,* and *Pediococcus* genera are those most frequently identified as epiphytes within the microbiota [14]. Although several species have been identified and characterized, the selection of new strains with valuable biotechnological

Ecuador is known as a megabiodiverse country, where access to genetic resources

Recently, we proposed to investigate the microbiota associated with wild fruits; especially, to select and characterize strains of beneficial lactic acid bacteria [16]. Tropical forest fruits provide essential nutrients, minerals, and antioxidants that keep a healthy body and provide resilience to disease [17]. The physical and

for research is still deficient. Studies on the microbial diversity of unexplored niches and environments are of interest as these might lead to the identification of an endless number of species with unique characteristics and properties. It has been claimed that the microbial population present in raw plant material that has been originated from unexplored niches, differs among samples, as the plant matrix might carry a particular microbiota in a specific geographical region and at a

**16**

#### **Figure 1.**

*Location of samples obtained for this study in the Amazon region of Ecuador. Colored areas represent national parks. Most of the samples were obtained in the areas adjacent to the Cuyabeno National Park.*

*Malus* sp., *Costus* sp., *Solanum stramofolium, Cheilocostus specious*, and *Theobroma grandiflorum* were collected aseptically in sterile bags and stored at 4°C for transport to the laboratory. The fruit samples were washed with a 5% bleach solution for 5 min, then twice with distillate water, smashed in small pieces, and transferred into Erlenmeyer flasks (500 ml) that contained peptone water (0.1%, bacteriological peptone, Difco Detroit, MI, USA). Samples were incubated statically for up to 5 days at room temperature. MRS agar [26] plates containing 5 g/l CaCO3 were inoculated and incubated under anaerobic conditions (37°C for 72 hours), and individual colonies were randomly selected and purified. The purified colonies (<75 colonies/each fruit) were Gram-stained, characterized by microscopic morphology, and tested for mobility, indole and catalase production, spore formation, and gas production from glucose [16]. The selected colonies were screened for their capacity to inhibit four bacterial pathogens (Section 2.4), resistance to bile (1%), acidity (pH 2.5-3.0), and temperature range (15-45°C). A total of 41 isolates complying with these criteria were further selected and identified. Each isolate was assigned a collection code (UTN) and was stored at −80°C in MRS medium containing 20% glycerol.

#### **2.2 Classification of isolates using 16S rRNA gene sequence analysis**

16S rRNA gene sequencing was used for taxonomical assignment (Macrogen Inc., Korea, custom service). The primers 27F 5′ (AGA GTT TGA TCM TGG CTC

**19**

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity…*

AG) 3′ and 1492R 5′ (TAC GGY TAC CTTGTT ACG ACT T) 3′ were used for the PCR amplification. The PCR reaction was performed with 20 ng of genomic DNA in a 30 μl reaction mixture with EF-Taq (SolGent, Korea) and through the following protocol: activation of Taq polymerase at 95°C for 2 minutes, 35 cycles of 95°C for 1 minute, at 55°C and 72°C for 1 minute, finishing with 10 minutes at 72°C. The amplification products were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). The sequencing reaction was performed using a PRISM BigDye Terminator v3.1 cycle sequencing kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA). The mixture was incubated at 95°C for 5 minutes, followed by 5 minutes

on ice and then analyzed by an ABI Prism 3730XL DNA analyzer (Applied

algorithm on the 16S ribosomal RNA database at NCBI, as implemented in Geneious Prime 2020.2.3 [27]. This first search was used to obtain a maximum of 100 hits and associated search quality parameters that provided an initial reference for taxonomic classification. A final taxonomic assignment was made by the RDP Bayesian classifier algorithm [28] with 100 bootstrap replicates and a K-mer of size 8, as implemented in the function "accurate, high-resolution sample inference from amplicon sequencing data" (assign Taxonomy) of the DADA2 package [29] in R [30]. The Genome Taxonomy Database [31] was used as the reference for the Bayesian classifier algorithm. A threshold for bootstrap values equal or over 80% was used to filter the taxonomic correspondences. The resulting final table of taxonomic assignments was used for downstream

We used reference samples to root the inferred phylogenetic hypotheses and provide a comparison to the experimental samples used in this study. This reference samples were: KJ660075.1: *Brachyspira\_hampsonii*; AJ276460.1: *Enterococcus\_faecalis;* NR\_114312.1: *Weissella oryzae*; NR\_104573.1: *Lactobacillus*; LC063164.1: *Weissella* 

LAB represent a small fraction of the autochthonous microbiota in the fruit and plant phyllosphere. However, due to the inherent limitations of in vitro cultures, the most frequent isolates often belong to the genera *Lactobacillus, Leuconostoc, Weissella, Enterococcus*, and *Pediococcus* [14]. More recently, based on conserved pairwise average amino acid identity, core genome phylogeny, physiological criteria, clade-specific signature genes, and ecology, *Lactobacillaceae*, and *Leuconostocaceae* were merged into *Lactobacillaceae* [32]. The genus *Lactobacillus* was split into 25 genera including *Lactiplantibacillus, Fructiplantibacillus, Lactobacillus*, among others. Thus, we allocate the study samples according to this new classification.

The BBL Crystal anaerobe identification system (cat # 245010, BD Company, US) is a miniaturized identification method for tests of fermentation, oxidation, degradation, and hydrolysis of diverse substrates. It also includes chromogen and fluorogenic linked substrates to detect enzymes that microbes metabolize. The results obtained after incubation of strips for 24 hours at 37°C were analyzed using the BBL Crystal analysis software (according to the manufacturer instructions). The results were qualitatively defined as negative (−) or positive (+) according to the detected color. As a reference for the metabolic profile test, we used *Lactobacillus plantarum* ATCC8014 and *Lactobacillus fermentum* CNCM 1-2998 (Lacc), which were recovered from the commercial probiotic Lacteol Fort

A preliminary fast search of the sequences was conducted using the megablast

*DOI: http://dx.doi.org/10.5772/intechopen.94179*

Biosystems, Foster City, CA).

statistical analyses.

*confusa;* NR\_029133.1: *Lactobacillus.*

**2.3 Metabolic profile of the selected isolates**

(*Lactobacillus* LB, Axcan Pharma, France).

*Microbiota of Wild Fruits from the Amazon Region of Ecuador: Linking Diversity… DOI: http://dx.doi.org/10.5772/intechopen.94179*

AG) 3′ and 1492R 5′ (TAC GGY TAC CTTGTT ACG ACT T) 3′ were used for the PCR amplification. The PCR reaction was performed with 20 ng of genomic DNA in a 30 μl reaction mixture with EF-Taq (SolGent, Korea) and through the following protocol: activation of Taq polymerase at 95°C for 2 minutes, 35 cycles of 95°C for 1 minute, at 55°C and 72°C for 1 minute, finishing with 10 minutes at 72°C. The amplification products were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). The sequencing reaction was performed using a PRISM BigDye Terminator v3.1 cycle sequencing kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA). The mixture was incubated at 95°C for 5 minutes, followed by 5 minutes on ice and then analyzed by an ABI Prism 3730XL DNA analyzer (Applied Biosystems, Foster City, CA).

A preliminary fast search of the sequences was conducted using the megablast algorithm on the 16S ribosomal RNA database at NCBI, as implemented in Geneious Prime 2020.2.3 [27]. This first search was used to obtain a maximum of 100 hits and associated search quality parameters that provided an initial reference for taxonomic classification. A final taxonomic assignment was made by the RDP Bayesian classifier algorithm [28] with 100 bootstrap replicates and a K-mer of size 8, as implemented in the function "accurate, high-resolution sample inference from amplicon sequencing data" (assign Taxonomy) of the DADA2 package [29] in R [30]. The Genome Taxonomy Database [31] was used as the reference for the Bayesian classifier algorithm. A threshold for bootstrap values equal or over 80% was used to filter the taxonomic correspondences. The resulting final table of taxonomic assignments was used for downstream statistical analyses.

We used reference samples to root the inferred phylogenetic hypotheses and provide a comparison to the experimental samples used in this study. This reference samples were: KJ660075.1: *Brachyspira\_hampsonii*; AJ276460.1: *Enterococcus\_faecalis;* NR\_114312.1: *Weissella oryzae*; NR\_104573.1: *Lactobacillus*; LC063164.1: *Weissella confusa;* NR\_029133.1: *Lactobacillus.*

LAB represent a small fraction of the autochthonous microbiota in the fruit and plant phyllosphere. However, due to the inherent limitations of in vitro cultures, the most frequent isolates often belong to the genera *Lactobacillus, Leuconostoc, Weissella, Enterococcus*, and *Pediococcus* [14]. More recently, based on conserved pairwise average amino acid identity, core genome phylogeny, physiological criteria, clade-specific signature genes, and ecology, *Lactobacillaceae*, and *Leuconostocaceae* were merged into *Lactobacillaceae* [32]. The genus *Lactobacillus* was split into 25 genera including *Lactiplantibacillus, Fructiplantibacillus, Lactobacillus*, among others. Thus, we allocate the study samples according to this new classification.

#### **2.3 Metabolic profile of the selected isolates**

The BBL Crystal anaerobe identification system (cat # 245010, BD Company, US) is a miniaturized identification method for tests of fermentation, oxidation, degradation, and hydrolysis of diverse substrates. It also includes chromogen and fluorogenic linked substrates to detect enzymes that microbes metabolize. The results obtained after incubation of strips for 24 hours at 37°C were analyzed using the BBL Crystal analysis software (according to the manufacturer instructions). The results were qualitatively defined as negative (−) or positive (+) according to the detected color. As a reference for the metabolic profile test, we used *Lactobacillus plantarum* ATCC8014 and *Lactobacillus fermentum* CNCM 1-2998 (Lacc), which were recovered from the commercial probiotic Lacteol Fort (*Lactobacillus* LB, Axcan Pharma, France).

*Ecosystem and Biodiversity of Amazonia*

*Malus* sp., *Costus* sp., *Solanum stramofolium, Cheilocostus specious*, and *Theobroma grandiflorum* were collected aseptically in sterile bags and stored at 4°C for transport to the laboratory. The fruit samples were washed with a 5% bleach solution for 5 min, then twice with distillate water, smashed in small pieces, and transferred into Erlenmeyer flasks (500 ml) that contained peptone water (0.1%, bacteriological peptone, Difco Detroit, MI, USA). Samples were incubated statically for up to 5 days at room temperature. MRS agar [26] plates containing 5 g/l CaCO3 were inoculated and incubated under anaerobic conditions (37°C for 72 hours), and individual colonies were randomly selected and purified. The purified colonies (<75 colonies/each fruit) were Gram-stained, characterized by microscopic morphology, and tested for mobility, indole and catalase production, spore formation, and gas production from glucose [16]. The selected colonies were screened for their capacity to inhibit four bacterial pathogens (Section 2.4), resistance to bile (1%), acidity (pH 2.5-3.0), and temperature range (15-45°C). A total of 41 isolates complying with these criteria were further selected and identified. Each isolate was assigned a collection code (UTN) and was stored at −80°C in MRS medium containing 20% glycerol.

*parks. Most of the samples were obtained in the areas adjacent to the Cuyabeno National Park.*

*Location of samples obtained for this study in the Amazon region of Ecuador. Colored areas represent national* 

**2.2 Classification of isolates using 16S rRNA gene sequence analysis**

16S rRNA gene sequencing was used for taxonomical assignment (Macrogen Inc., Korea, custom service). The primers 27F 5′ (AGA GTT TGA TCM TGG CTC

**18**

**Figure 1.**

## **2.4 Antibiotic susceptibility testing**

Susceptibility to several antibiotics was determined by commercial discs of Amoxicillin (AMX: 25 μg), Ampicillin (AM: 10 μg) Gentamicin (CN: 10 μg), Kanamycin (K: 30 μg), Amoxicillin/Clavulanic Acid (AMC: 20/10 μg), Tetracycline (TE: 30 μg), and Cefuroxime (CXM: 30 μg). For the disk diffusion assay, we used concentrations recommended by the Scientific Committee on Animal Nutrition (discs provided by Merck) [12]. We used *Escherichia coli* ATCC 25922 as quality control. The microbiological breakpoints reported by the FEEDAP standards were used to categorize lactobacilli as susceptible or resistant [4]. *Lactobacillus plantarum* ATCC8014 and *L. fermentum* CNCM 1-2998 (Lacc) were used as a reference to the test. Results were qualified as R (resistance), I (intermediate), and S (susceptible).
