Full Text:The complex interaction between microbial community and food has long captivated researchers and gained substantial attention in the scientific community. Microbes are ubiquitous in the realm of foods and beverages, playing multifaceted roles and serving diverse functions that extend beyond their mere existence [1]. From contributing to shaping the sensory and health attributes to presenting a potential source of pathogenic threats, these microorganisms are key determinants of the quality, safety, and nutritional attributes of the edibles we consume [2, 3]. This emphasizes the necessity for a comprehensive understanding of microbial taxonomy and diversity within foods and beverages. It becomes all the more imperative when considering foods that are inherently prone to microbial colonization, given the direct interaction between food-associated microbes and the human host through the digestive system. These microorganisms can have a profound impact on human health, either positively, by playing essential roles in digestion, nutrient absorption, and overall well-being, or negatively, when potentially harmful strains pose inherent health risks [2, 4]. Therefore, the association between food and microbes necessitates comprehensive examination, not only for the maintenance of food quality but also to ensure the well-being of consumers.
Among the many consumable products, date palm sap, also known as "date palm juice," "date palm nectar," or "date tree sap," holds significant cultural importance and popularity, especially in regions where date palms grow [5]. This sweet nectar, collected directly from date palm trees, carries a rich heritage and is a cherished source of refreshment and nutrition. For centuries, it has been valued for its remarkable nutritional content, offering an abundance of vitamins, minerals, and natural sugars, making it a natural energy booster. Its sweetness and pleasant taste make it a popular choice for refreshing drinks and culinary applications, especially in Middle Eastern, South Asian and North African cuisines. Moreover, the sap is further processed into popular food-products such as date sap jaggery or sugar, highlighting its versatility in traditional and contemporary cuisine. Additionally, date palm sap also represents a microcosm of microbial diversity that remains relatively unraveled. But exploring bacterial composition within date tree sap can offer valuable insights into the preservation, flavor, and safety of this cherished beverage. By identifying the bacterial species present in date palm juice and analyzing their composition and diversity, it is possible to gain a deeper understanding of the interplay between microorganisms and this beverage, ultimately affecting its safety and quality.
Metagenomics, with its capability to unveil the genetic blueprints of entire microbial communities, has revolutionized the study of bacterial diversity in foods. This powerful approach transcends traditional culture-dependent methods, allowing researchers to identify and analyze the diversity and taxonomy of microbial communities directly from environmental samples [6]. Metagenomic analysis offers a comprehensive view of the microbial inhabitants in the ecosytem, enabling precise characterization of bacterial taxonomy, their abundance, community dynamics, and how they interact in a given environment [7]. Its high-throughput capabilities, affordability, and adaptability to specific research objectives make it an indispensable tool for tracking changes of microbial communities over time and understanding their ecological impacts, thus enhancing our understanding of the microbial realm.
In the present study, we employed metagenomic approach to explore the bacterial diversity, taxonomy, abundance, and community structure present within date tree sap. This research contributes to our understanding of the intricate microbial ecosystem inherent to this widely consumed beverage. By elucidating the microbial composition of date tree sap, our findings emphasizes its importance for broader implications in quality control, safety assurance, potential health benefits .
Four sap samples were collected from four different date palm trees in Sitakund Upazila. The samples were carefully collected in sterile tubes, placed in a cooler box, and transported to the laboratory.
DNA extraction and polymerase chain reaction (PCR)
DNA extraction from the samples was performed, according to a modified version of the protocol described by Han et al. (2018) [8] in BD Genomes, Bangladesh. Following centrifugation, the pellet was resuspended in extraction buffer with lysozyme and incubated at 60°C for 30 minutes, followed by cooling to room temperature. RNAse A was then added and the mixture incubated at 30°C for another 30 minutes. Subsequently, proteinase K was added, and the samples were incubated in a water bath at 65°C for 60 minutes, with inversion every 20 minutes. DNA extraction was carried out using the phenol-chloroform method, followed by resuspension in 100 µL of TE buffer and heating at 50°C for 10 minutes prior to storage at -20°C. DNA quality was evaluated on a 1% agarose gel, and the DNA concentration was measured using a Nanodrop followed by an Agilent 5400. The V3-V4 region of the 16S rRNA gene was amplified using the primer set 515F and 806R with barcodes (515F: GTGCCAGCMGCCGCGGTAA and 806R: GGACTACHVGGGTWTCTAAT). All PCR reactions were performed with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM of forward and reverse primers, and approximately 10 ng of template DNA. The thermal cycling protocol included initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, with a final extension step at 72°C for 5 min. After amplification, PCR products were mixed with an equal volume of 1× loading buffer containing SYBR Green and subjected to electrophoresis on a 2% agarose gel for visualization. The PCR products were then purified using the Qiagen Gel Extraction Kit (Qiagen, Germany).Top of Form
Library preparation, sequencing and quality control
The amplicon metagenomic analysis was conducted at Novogene Company, China arranged via BD Genomes. Sequencing libraries were prepared using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer's protocol, and index codes were incorporated. The library quality was assessed using the Qubit® 2.0 Fluorometer (Thermo Scientific) and the Agilent Bioanalyzer 2100 system. Subsequently, the libraries were sequenced on an Illumina NovaSeq 6000 platform (PE250), generating 250x2 bp paired-end reads for the V3-V4 regions of the 16S rRNA gene. Paired-end reads were assigned to samples based on their unique barcodes and trimmed by removing the barcode and primer sequence. The paired-end reads were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) [9]. The merged sequences were referred to as raw tags. Quality filtering was applied to the raw tags under specific conditions to obtain high-quality clean tags [10], following the QIIME2 (V1.9.1) pipeline [11]. The clean tags were compared with the reference Silva database [12] using the UCHIME algorithm [13] to identify chimeric sequences, which were subsequently filtered out [14]. Finally, the effective tags were obtained.
Data filtering and analysis
The filtered data were denoised through DADA2, and sequences with an abundance of less than 5 were filtered out to yield the final ASVs. Species annotation was performed on each ASV's representative sequence to acquire species information and abundance distribution. Further analyses included abundance and alpha diversity to assess species richness and evenness within the sample, and to identify common and unique ASVs among different samples or groups [15].
The metagenome-based functional prediction of the microbial community present in the date sap samples was conducted using the PICRUSt2 software. Key functional processes were inferred based on the Kyoto Encyclopedia of Genes and Genome (KEGG) database [16].
Metagenomic sequence analysis of the date palm sap microbiota
The bacteriome associated with date palm tree sap was characterized through metagenomic investigations, employing sequencing of the V3-V4 region of the 16S rRNA gene. Four freshly collected samples (designated as S1, S2, S4, and S5) from different date palm trees were subjected to metagenomic analysis to determine their microbial compositions. The pH of the samples fell within the acidic range, measuring 3.98, 4.15, 4.35, and 4.80, respectively. This acidity likely results from the fermentation of sugars by various bacteria present in the sap.
The metagenomic sequencing of the four samples generated a total of 447,165 raw reads. These reads were assembled into 438,499 combined reads, followed by the removal of low-quality and short reads, resulting in 425,316 qualified data. Subsequently, chimera sequences were filtered out, yielding 361,759 effective sequences with an average read length of 422 bp (Table 1). On average, 90,440 non-chimeric high-quality reads were obtained from 111,791 raw reads.
The “good coverage of library” of the metagenomics data was >99.9% for all samples indicating almost complete sampling of the species present. Jaccard distance matrix revealed a high level of similarity among the samples (ranging from 68% to 98%, as shown in Figure 1), suggesting a common bacterial community shared across a significant portion of the date palm sap samples.
Table 1. Read processing and metrics for the metagenomic data of date palm juice microbiota. Read counts before and after merging and quality filtering are presented.
Figure 1. 16S-metagenomic analysis of bacterial communities in date tree sap. Jaccard similarity index, computed from the metagenomic data, indicates similarity levels between bacterial communities in the raw sap samples. Darker color corresponds to higher similarity.
Bacterial community-structure and diversity in date palm sap
Ninety seven percent of the effective sequences were assembled into ASVs through DADA2 to discern the community composition and diversity of microbes present in the date sap samples. In total, 2,572 distinct ASVs were generated at the kingdom level from the four samples, with 99.9% attributed to bacteria and the remaining comprising archaea and others. Taxonomic annotation, performed at a 97% similarity level, categorized the ASVs into a total of 38 phyla, 92 classes, 213 orders, 352 families and 686 genera.
The microbial community dynamics revealed that certain bacterial taxa were consistently present across all samples dominating the sap microbiome at each taxonomic level. The relative abundance of the taxonomic groups at various hierarchical levels, from phylum to genus, is presented in Figure 2. At the phylum level, Proteobacteria and Firmicutes were the most abundant, with average relative abundances of approximately 49% and 40%, respectively, consistently prevalent across all samples. Although variations were observed in their relative abundances, these two phyla collectively constituted a substantial proportion of the total microbial community, ranging from 75.32% to 98.4% in different samples. Additionally, other phyla, including Actinobacteria, Bacteroidetes, Campylobacterota, and Desulfobacterota, were also detected in all samples although at relatively lower levels, with Campylobacterota notably absent in one sample. At the order level, four taxa including Lactobacillales, Sphingomonadales, Pseudomonadales, Burkholderiales were abundantly present in the various sap samples.
At the genus level, Leuconostoc emerged as the most prominent group, representing approximately 20% of the microbial community on average, followed by Zymomonas (~13%) and Lactobacillus (~11%). These genera exhibited varying degrees of dominance among the samples, underscoring the complex microbial landscape of date palm sap. We also identified additional genera in the date tree sap, including Acinetobacter, Fructobacillus, Lactococcus, Pseudomonas, Sphingomonas, Ralstonia, and Thauera, which were relatively less dominant but common in all samples. Some of the low abundant genera, however, was not shared across the samples, such as Rothia, C39, Pseudarcobacter, Ralstonia, Marinomonas, Novosphingobium, and others.
Figure 2. Metagenomic insights into the diversity and abundance of bacterial communities in date palm sap at various taxonomic hierarchies. The heatmaps represent the taxon’s abundance distribution, with the color codes indicating various abundance levels. The 20 dominant groups are presented at each taxonomic position, revealing Proteobacteria and Firmicutes as the predominant phyla in the microbiota of raw palm sap, while Leuconostoc, Lactobacillus, and Zymomonas were most abundant at the genus level.
Figure 3. Metagenome-predicted functions of the bacterial community in date palm sap. Functional predictions of the sap microbiota were generated using PICRUSt2 against the KEGG database, revealing key functional roles categorized into major cellular pathways and processes at KEGG levels 1 (top) and 2 (bottom).
Functional prediction of the date palm sap microbiota
The metagenome-predicted function of the sap bacterial community was inferred using PICRUSt matched against the KEGG database [17]. The functional prediction indicated a diverse array of functional roles for the bacteriome associated with various cellular pathways, bioprocesses and systems (Figure 3) with the majority of the reads involved in metabolic processes accounting for about half of the total (48.7%) in the four samples, followed by genetic information processing (17%), environmental information processing (13%), and others at KEGG level 1. Further at level 2, each of these cellular processes were distributed into more specific functions. For example, the metabolic activities were mostly attributed to amino acid metabolism (20.7%), carbohydrate metabolism (19.8%), energy metabolism (11.3%), and so forth. On the other hand, replication and repair was inferred to be responsible for a large proportion (44.8%) of genetic information processing, followed by translation (29%), folding, sorting and degradation (13.2%), and transcription (13%). Additionally, membrane transport emerged as the predominant function (82.1%) within environmental information processing, followed by signal transduction (16.5%) and signaling molecules and interaction (1.38%).
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This study identified the bacterial taxa that shape microbial landscape of date palm sap. The most dominant microbial groups that have been identified were common in all samples, suggesting that they play a central and consistent role in defining the core microbiota of date palm sap. At the phylum level, the dominance of Proteobacteria and Firmicutes aligns with the findings of previous reports on various fermented foods and beverages, where these phyla often play pivotal roles in the fermentation process. For example, Proteobacteria have been associated with the initial stages of fermentation, converting sugars into organic acids, while Firmicutes often play vital roles in later fermentation stages, enhancing flavor and texture [18]. In this context, the dominance of the genera Leuconostoc, Zymomonas, and Lactobacillus in date palm sap is also noteworthy, as these genera are recognized for their significant roles in the fermentation of plant-based beverages, often influencing taste, aroma, and overall quality [19–21]. Leuconostoc and Lactobacillus are lactic acid bacteria (LAB), which are often responsible for the production of organic acids, such as lactic acid, leading to a tangy taste, enhanced acidity and preservation [22]. This aligns with our findings, as the pH of all date palm sap samples fell within the range of approximately 4 to 5. Furthermore, many of the LAB are also considered beneficial and have potential as probiotics, offering advantages such as enhancing the nutritional value of fermented foods, improved digestion, and potential immune system support [23, 24]. Zymomonas, the other prominent palm sap genus, is involved in the conversion of sugars to alcohol and carbon dioxide, which can influence the alcoholic content and effervescence of the beverage [25]. These genera potentially play critical roles in shaping the sensory attributes and quality of date palm sap. The dominance of these genera in date palm sap can be attributed to the sugar-rich environment of the sap, as it contains high levels of various sugars including glucose, sucrose, and fructose. This suggests that the sugar content of date palm sap provides a favorable habitat for these bacteria, enabling them to thrive and influence the beverage's quality and taste.
The diverse bacterial taxa identified in date palm sap exhibit distinct ecological niches and occurrences, aligning with their known habitats. Leuconostoc and Zymomonas are commonly found in sugar-rich environments and play crucial roles in the fermentation of plant-based beverages [26]. Acinetobacter, Pseudomonas, and Rothia are versatile bacteria with a wide occurrence in various water-rich settings and have been noted for their metabolic adaptability [27–29]. Fructobacillus, known for its preference for fructose-rich environments, is often associated with the fermentation of fruits. Lactococcus, a lactic acid bacterium, thrives in dairy products and can contribute to the fermentation process [30]. C39, Pseudarcobacter, and Ralstonia, although less commonly studied, are often encountered in diverse environmental niches, with potential contributions to various bioprocesses [31]. Marinomonas, Methyloversatilis, and Novosphingobium are typically found in aquatic environments, showcasing their adaptability to water-rich habitats [32–34]. Marivita and Hydrogenophaga are associated with water ecosystems [35], potentially playing essential roles in nutrient cycling. Flavobacterium, known for its widespread distribution, often occurs in aquatic habitats, contributing to organic matter decomposition and nutrient cycling [36]. Candidatus Aquiluna is typically observed in marine environments, where it may play a role in marine nutrient cycling [37]. These observations highlight the diverse ecological roles and adaptability of these genera, contributing to the complex microbial ecology of date palm sap.
However, the presence of the common genera, such as Acinetobacter and Pseudomonas, also raises concerns about their potential effects on the beverage, including the risk of spoilage. These genera have been associated with various metabolic pathways, including the breakdown of organic compounds and the potential production of off-flavors or spoilage compounds [38]. The presence of these genera should be monitored, as their metabolic activities could affect the sensory attributes and safety of the beverage.
It is essential to emphasize that the specific interactions and roles of these bacterial groups in date palm sap require further investigation to understand their full implications for the quality, safety, and potential health benefits of this culturally cherished beverage. For example, the absence of Campylobacterota in one sample, while moderately abundant in others, underscores the microbial diversity within a seemingly uniform product. This variation highlights the need for continued research into the microbial ecology of date palm sap. Moreover, our metagenomic analysis was conducted on a relatively limited scale encompassing four different date tree sap samples. While this approach provided valuable insights, a more comprehensive understanding of microbial diversity and dynamics in date palm sap could be achieved by including a larger number of samples from various geographical regions and countries, and ensuring robust analysis for each sample. Nonetheless, the findings of our study contribute significantly to our initial understanding of the microbial composition within date palm sap, laying a foundation for further research aimed at refining the quality and safety of this valued beverage.
Looking ahead, future research endeavors can build upon this foundation by conducting extensive analyses of microbial diversity incorporating samples sourced from various regions worldwide. This broader approach will offer a global perspective on bacterial composition in date palm sap, enriching our knowledge and paving the way for comprehensive improvements in the production and safety protocols of this culturally significant beverage. Furthermore, future studies should prioritize the isolation of key bacterial taxa to uncover and harness their precise contributions to specific juice characteristics, ensuring a comprehensive understanding and practical application of the microbial diversity within date palm sap.
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Funding
This study was supported by a grant from the Grant for Advanced Research in Education, Bangladesh Bureau of Educational Information and Statistics, Ministry of Education, Government of the People's Republic of Bangladesh (Ref: LS20201294).
Conflict of interest
The authors have no conflicts of interest to declare.
Ethical approval
This article does not contain any experiments done with human participants or animals performed by any of the authors.
Acknowledgment
The authors would like to express their gratitude to Mohammad Nazmul Ahmed Chowdhury for facilitating communication with the date tree owners. The authors also extend their appreciation to the members of the Laboratory for Health, Omics, and Pathway Exploration (HOPE Lab), Chattogram, Bangladesh for their assistance in this project.
Author contributions
TJH conceived the idea and secured funding for this project. TJH and SH designed the study. RI, IHN and MSK collected and managed samples. SH performed data processing and bioinformatics study. SH and TJH contributed to data interpretations. TJH drafted the manuscript and SH wrote the methods related to the metagenomic analysis. All authors read and approved the submitted manuscript.
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