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Faculty of Electrical Engineering, Mathematics and Computer Science (EEMCS), University of Twente, Enschede, 7522 NB, the NetherlandsResearch Centre on Interactive Media, Smart Systems and Emerging Technologies–RISE, Nicosia 1066, Cyprus
The interest in milk originating from donkeys is growing worldwide due to its claimed functional and nutritional properties, especially for sensitive population groups, such as infants with cow milk protein allergy. The current study aimed to assess the microbiological quality of donkey milk produced in a donkey farm in Cyprus using culture-based and high-throughput sequencing techniques. The culture-based microbiological analysis showed very low microbial counts, whereas important food-borne pathogens were not detected in any sample. In addition, high-throughput sequencing was applied to characterize the bacterial communities of donkey milk samples. Donkey milk mostly composed of gram-negative Proteobacteria, including Sphingomonas, Pseudomonas, Mesorhizobium, and Acinetobacter; lactic acid bacteria, including Lactobacillus and Streptococcus; the endospores forming Clostridium; and the environmental genera Flavobacterium and Ralstonia, detected in lower relative abundances. The results of the study support existing findings that donkey milk contains mostly gram-negative bacteria. Moreover, it raises questions regarding the contribution of (1) antimicrobial agents (i.e., lysozyme, peptides) in shaping the microbial communities and (2) bacterial microbiota to the functional value of donkey milk.
Milk from nontraditional animal species (i.e., donkey, camel, and buffalo) are recently gaining interest for research and regulatory authorities, mainly because they are considered valuable alternative nutritional sources for specific population groups (i.e., infants, the elderly, immunocompromised, and those allergic to cow milk protein;
). In particular, the interest in donkey milk has increased dramatically over the past few years due to its nutritional, nutraceutical, functional, and immunological properties (
Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice.
Donkey milk is characterized by a very low microbial population, which can be attributed to the increased concentrations of antimicrobial factors, including lysozyme and lactoferrin (
). Lysozyme is an enzyme that catalyzes the hydrolysis of glycosidic bonds between N-acetylmuramic acid and N-acetyl-d-glucosamine residues of peptidoglycan, the primary component of the bacterial cell wall (
); (3) binds additional substances and compounds, including heparin, DNA, glycosaminoglycans, as well as metal ions such as Mn3+, Al3+, Co3+, Ga3+, Zn2+, Cu2+, and so on (
). Indeed, the microbiological data of raw donkey milk show a significantly low total bacteria count with a mean population of 2.40 to 5.87 log cfu/mL (
). However, the presence of food-borne pathogens such as Escherichia coli O157, Listeria monocytogenes, Bacillus cereus, and Campylobacter spp., have been detected in some studies (
Hygienic characteristics and microbiological hazard identification in horse and donkey raw milk (Caratteristiche igienico-sanitarie ed identificazione dei rischi microbiologici nel latte di cavalla e di asina).
Despite the recognized benefits of donkey milk consumption, the existing microbial consortia, and their possible contribution to the milk's nutritional value, have not been evaluated yet. Although various culture-dependent methodologies have identified the presence of bacteria, including food-borne pathogens, in aseptically collected milk, they do not suffice to provide complete information regarding several additional genera present in low numbers or difficult to be cultured (
). Recently, the high-throughput sequencing (HTS) technology has been applied for deeper identification of the vastly diverse bacterial communities present in different types of milk (
). This technology provides the ability to characterize the microbiota present within a sample comprehensively, and it is characterized by increased sensitivity and high throughputness in comparison to other culture-independent methodologies. Amplicon sequencing achieves this by generating and sequencing in parallel thousands of specific DNA sequences, such as the bacterial 16S rDNA gene (
). The microbiome of donkey milk is hypothesized to be composed of bacteria commonly found in milk samples, but with adaptation to the elevated presence of antimicrobial compounds. Previous HTS studies on donkey milk bacterial communities identified increased relative representation of gram-negative bacteria, such as Pseudomonas spp. (
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
Therefore, considering the growing interest in donkey milk for infants, adults, and elderly, the study aims to identify and characterize the bacterial communities of donkey milk produced in a donkey farm in Cyprus, as well as to evaluate its microbiological quality by using culture-based approaches in combination with Illumina MiSeq (Illumina, San Diego, CA) amplicon sequencing. The extracted findings are expected to increase knowledge regarding the bacterial consortium comprising the donkey milk and provide indications of the key bacterial microbiome that contributes to donkey milk's elevated nutritional value.
MATERIALS AND METHODS
Collection of Milk Samples
Milk samples were collected from the Golden Donkeys Farm, located in Larnaca District, Cyprus. All donkeys were fed on the same diet consisting of hay, barley, corn, and a concentrate of minerals, vitamins, and salt following the European Directive 98/58/EC (European Union, 1998). Donkeys were healthy and no antibiotics were administered before sampling. Milking was carried out in the stable and donkeys were milked manually from the same milker, adhering to strict personal hygiene conditions, once daily during the morning. Milk was immediately transferred to refrigeration (6°C) until further processing (same day).
Sampling for chemical and microbiological analysis was conducted weekly (33 wk) from October of 2018 to May 2019 from the daily milk batch (20 L from 20 to 25 milking donkeys). Milk samples for 16S rRNA gene amplicon analysis was conducted in May 2019 from 11 donkeys. For chemical and microbiological analysis from each donkey, a total of 250 mL of milk from both mammary glands were collected into a 250-mL sterile container. For 16S rRNA gene amplicon analysis a total of 100 mL of milk from both mammary glands was collected into two 50-mL sterile tubes (2 samples/donkey). During milking, the udder was cleaned using sterile wet wipes and the nipples using 70% ethanol and dried with sterile gauze. The donkeys were all multiparous. Donkey milk samples were placed in cool-boxes and immediately transported to the laboratory, where (1) they were kept at 4°C and processed during the same working day for chemical and microbiological culture-based analysis, or (2) stored at −80°C for 16S rRNA gene amplicon-HTS analysis.
Chemical Analysis and Lysozyme Activity
Chemical analyses of fresh raw donkey milk were performed by using standard methods [i.e., total nitrogen content (ISO 8968-1:2014,
)]. All the analyses were done in triplicates, and average values were reported. Lysozyme concentration was quantified using an ELISA kit (Human Lysozyme ELISA KIT ab108880; Abcam, Cambridge, UK). The test was performed according to the manufacturer's instructions and the absorbance was determined spectrophotometrically (Infinite PRO 200, Tecan, Switzerland), at 450 and 570 nm. All samples were analyzed in triplicate.
Microbiological Analysis
All samples were evaluated for total aerobic bacteria, Enterobacteriaceae, staphylococci, Escherichia coli, and Bacillus cereus after serial dilutions in saline solution (0.85% wt/vol), using pour or spread plate technique. Table 1 shows the growth media, incubation time, temperature, and specific method used for each group of microorganisms inspected. Listeria monocytogenes analysis performed by using ISO method 11290–1:2017 (
ISO 11290-1:2017. Microbiology of the food chain—Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. Part 1: Detection method..
International Organization for Standardization,
Geneva, Switzerland2017
PCA = plate count agar; VRBGA = violet red bile glucose agar; BP = Baird Parker; MYP = mannitol egg yolk polymixin; TBX = tryptone bile x-glucuronide; ALOA = Listeria chromogenic agar base acc. to Ottaviani and Agosti.
1 PCA = plate count agar; VRBGA = violet red bile glucose agar; BP = Baird Parker; MYP = mannitol egg yolk polymixin; TBX = tryptone bile x-glucuronide; ALOA = Listeria chromogenic agar base acc. to Ottaviani and Agosti.
ISO 4833-2:2013. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of microorganisms—Colony-count technique at 30 degrees C by the surface plating technique..
International Organization for Standardization,
Geneva, Switzerland2013
ISO 21528-2:2004. Microbiology of food and animal feeding stuffs—Horizontal methods for the detection and enumeration of Enterobacteriaceae—Part 2: Colony-count method..
International Organization for Standardization,
Geneva, Switzerland2004
ISO 6888-1(1999). Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium..
International Organization for Standardization,
Geneva, Switzerland1999
ISO 7932:2004. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of presumptive Bacillus cereus—Colony-count technique at 30 degrees C..
International Organization for Standardization,
Geneva, Switzerland2004
ISO 16649-2:2001. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli—Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide..
International Organization for Standardization,
Geneva, Switzerland2001
ISO 11290-1:2017. Microbiology of the food chain—Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. Part 1: Detection method..
International Organization for Standardization,
Geneva, Switzerland2017
Five milliliters of donkey milk was mixed with 45 mL of 2% tri-sodium citrate (Honeywell, Charlotte, NC). After centrifugation at 16,000 × g for 5 min at 4°C, the top fat layer was removed using sterile cotton swabs, based on the manufacturer's instructions for the MoBio Microbial Kit (MoBio Laboratories Inc., Carlsbad, CA; https://www.selectscience.net/products/powerfood-microbial-dna-isolation-kit/?prodID=85256). The isolated DNA was kept at −20°C until processing.
Quantification of Total DNA
The total DNA extracted from the donkey milk samples was quantified fluorometrically using Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA) and Qubit dsDNA HS Assay Kit (Invitrogen). Evaluation of DNA purity achieved by measuring the ratios of absorbance A260/280 nm and A260/230 nm, using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, Waltham, MA).
Barcoded Illumina MiSeq Amplicon Sequencing of Bacterial 16S rRNA Gene
A HTS approach was applied to isolated donkey milk DNA for the identification of the existing bacterial communities. The bacterial V3–V4 hypervariable region of the 16S rDNA gene was amplified with the following 16S rDNA gene amplicon PCR primer pairs: (1) forward primer (CCTACGGGNGGCWGCAG) and (2) reverse primer (GACTACHVGGGTATCTAATCC), with the overhang adapter sequence addition. The paired-end approach based on the Illumina's protocol was applied (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) and as described by
). The quantification of each PCR product DNA concentration was performed using Qubit dsDNA High sensitivity assay. The estimation of DNA quality was evaluated using a bioanalyzer (Agilent 2200 TapeStation, Santa Clara, CA; expected size ~550 bp). The purification of each PCR amplicon was performed using NucleoMag NGS Clean-up and Size Select (Macherey-Nagel, Düren, Germany). Total amplicon products were normalized in equal concentrations and mixed in a single tube. The MiSeq 300 cycle Reagent Kit v2 (Illumina; 5% PhiX) was applied for the sequencing runs, whereas the sequencing reaction was performed on a MiSeq Illumina sequencing platform.
Bacterial Microbiome and Statistical Analysis
The FASTQ sequences were analyzed using Qiime 2 version 2020.2 (
). For quality filtering of raw reads, the Phred33 quality threshold was applied. Adapter sequence removal, FASTQ trimming, and read quality control performed using Trimmomatic (
) performed correction of Illumina-sequenced amplicon errors, discarding reads with undesired quality and with more than 2 expected errors, as well as removing chimeric sequences. Sequences were aligned using Mafft (via q2-alignment;
). Alpha rarefaction analysis, α diversity metrics (Faith's phylogenetic diversity, Shannon, inverse Simpson, and Chao1), and β diversity index (Bray-Curtis similarity) were evaluated via the Qiime2 (version 2020.2) and primer e v7 (https://www.primer-e.com). Principal coordinate analysis was estimated using q2-diversity after 11 samples were rarefied (subsampled without replacement) to 77,143 sequences per sample. Alpha rarefaction curve was plotted with 25 sampling depths. The clustering of the 16S rDNA sequences and the filtering in operational taxonomic units (OTU) was performed using 16S Metagenomics App from BaseSpace against the Illumina-curated version of GreenGenes (v.05.2013;
). The classified OTU were defined at ≥97% of sequence homology and converted to percentages (relative abundances), to determine the representation of each microbe among treatments. The OTU with relative abundance lower than 0.001% were excluded.
All raw sequence data in read-pairs format were deposited to the National Centre for Biotechnology Information in the Sequence Read Archive under BioProject PRJNA612663.
RESULTS
Chemical Analysis
The chemical characteristics of fresh raw donkey milk were evaluated for the period of October 2018 to May 2019, and results are presented in Table 2. Raw donkey milk was characterized by a mean protein content around 1.62 g/100 mL and a mean fat content of 0.84 g/100 mL. The mean DM observed in the current donkey milk study was 9.23 g/100 mL. The mean lysozyme concentration was 2.9 ± 0.9 mg/mL.
Table 2Chemical analysis of donkey milk samples (n = 33)
Table 3 presents the microbiological results of the 33 raw donkey milk samples for total viable microorganisms, staphylococci, Enterobacteriaceae, Escherichia coli, Bacillus cereus, and Listeria monocytogenes. The mean value of viable microorganisms was 3.80 log10 cfu/mL. Furthermore, staphylococci and Enterobacteriaceae were less than 4.7 and 3.4 log10 cfu/mL, respectively, whereas Escherichia coli, Bacillus cereus, and Listeria monocytogenes were not detected.
Table 3Microbiological quality of donkey milk samples (n = 33)
Abundance and Diversity of Members of the Bacterial Microbiota.
Eleven examined sample sets were used as input to the Illumina MiSeq to generate 281,294 high-quality sequencing reads, with an average of 25,572.18 sequencing reads per sample (range = 17,413–35,159, SD = 5,454.01) at the genus level (Table 4). High-quality sequences were grouped into average number 357.91 OTU (range = 266–492, SD = 66.35). Shannon, inverse Simpson, Chao1, and Chao2 estimators for genus level are also shown in Table 4.
Table 4Sample information, microbial diversity, and sequence abundance in genus level
Moreover, to evaluate differences in the bacterial community compositions of donkey milk samples, weighted UniFrac distance-based microbiota structure analysis was performed. Bray-Curtis similarity index indicated increased similarity among the bacterial communities of milk samples at genus level (Supplemental Table S1, https://doi.org/10.3168/jds.2020-19242). Principal coordinate analysis of Bray-Curtis distance indicated no effective discrimination between samples (Figure 1). The principal coordinates 1, 2, and 3 explained 62.24%, 10.30%, and 6.8% of the variation, respectively. The OTU network showed relation with changes in the microbial population and one main cluster was observed, from which the samples D6, D9, and D10 were discriminated.
Figure 1Principal coordinate analysis of donkey milk samples by plots of Bray-Curtis distance. Clustering of points means similarity in relative abundances of operational taxonomic units among those samples.
Taxonomic Composition of Bacterial Communities in Donkey Milk Samples.
According to 16S rDNA sequencing, the bacterial communities of donkey milk consisted of mostly members of the phylum Proteobacteria. Members of the phyla Firmicutes, along with Bacteroidetes, Actinobacteria, Acidobacteria, Cyanobacteria, and Verrucomicrobia, were detected in lower relative abundances. Figure 2 illustrates the bacterial composition of the donkey milk samples based on the percentage of sequence reads identified at the genus levels. The most commonly detected bacteria, identified in percentages greater than 1% in all analyzed samples, were the gram-negative bacteria Sphingomonas (16–47%), Pseudomonas (8–17%), and Mesorhizobium (11–25%), as well as the genus Acinetobacter, which was detected in increased relative abundances in 2 samples (samples G3 and G6: 24% and 16%, respectively). Moreover, Lactic acid bacteria (LAB), including the genera Lactobacillus and Streptococcus, were detected in relative abundances ranging from 1% to 4% in all samples tested. Additional commonly detected genera, but in reduced relative abundance, included the genera Ralstonia (0.02% to 2.6%), Aquabacter (0 to 5%), and its phylogenetically related Xanthobacter (0% to 5.5%), as well as the proteolytic Flavobacterium (0 to 5%). Furthermore, the number of reads representing 0.1% to 2% of the total reads per sample of the spore-forming, butyrate-producing Clostridium was also present.
Figure 2Three-dimensional 100% stacked column chart of the relative abundance of the major taxonomic groups detected by high-throughput sequencing at genus levels for 11 donkey milk samples, D1 to D11. Only operational taxonomic units with an incidence above 1% in at least 2 samples are shown.
The current study is the first report in which HTS technology applied to investigate the bacterial communities of Cyprus donkey milk. The 16S rRNA gene amplicon-HTS was used for an in-depth quantitative description of the bacterial population structure. Due to new information arising in recent years on the beneficial role of donkey milk consumption, such facilities are on the rise and milk production from other milk-producing species is becoming a niche product.
The results of the chemical composition of donkey milk samples are in line with other studies (
). The low content of donkey milk in fat is the main limitation for its use as the sole milk to children allergic to cow milk protein during their first year of life since recommended dietary targets may not be achieved unless adequately supplemented with medium-chain triglycerides (
). In most studies, including this one, low bacteria counts have been observed. Moreover, only a few studies have shown the presence of some food-borne pathogens, but in the present study, no food-borne pathogens were detected. Furthermore, the low total population of viable microorganisms complies with the EC Regulation 853/2004 (
), allowing the sale of donkey milk under the clause “other milk-producing species,” where the total bacterial plate count is less than 1,500,000 cfu/mL at 30°C. Noteworthy, if raw milk from species other than cows is intended for the manufacture of products made with raw milk by a process that does not involve any heat treatment, food business operators must take steps to ensure that the raw milk used meets the following criterion: plate count at 30°C (per mL) ≤500,000 (EC Regulation 853/2004;
). The high content of donkey milk in antimicrobial proteins, including lysozyme and lactoferrin, in combination with lactoperoxidase and immunoglobulins, are considered responsible for the low total bacterial counts (
The OTU analysis of the 16S rDNA gene sequences indicated that the gram-negative bacteria Sphingomonas, Mesorhizobium, and Pseudomonas were the most dominant genera detected in the Cyprus donkey milk samples. Other genera commonly occurred include Acinetobacter, Lactobacillus, Streptococcus, Ralstonia, Clostridium, and Flavobacterium. Previous metagenomic studies in donkey milk microbiota have also detected the presence of these genera, except Clostridium, but in different relative abundances (Table 5;
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
) indicated that the predominant genera were Pseudomonas, Ralstonia, Sphingobacterium, Acinetobacter, Cupriavidus, and Citrobacter, although the core bacterial representation differed among samples. This is probably because the samples obtained from 5 different donkey dairy farms during 2 yr, in contrast to the current study in which samples were obtained from one farm, during a shorter period. In agreement,
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
) identified also increased relative representation of the genus Pseudomonas in fresh donkey milk samples. Additional genera that detected in lower relative abundances included Chryseobacterium, Sphingobacterium, Stenotrophomonas, Citrobacter, and Delftia. Similar 16S rDNA sequencing analyses in human and bovine milk also identified the frequent presence of the genera Ralstonia, Sphingomonas, and Pseudomonas in all samples tested (
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
Human milk microbial community structure is relatively stable and related to variations in macronutrient and micronutrient intakes in healthy lactating women.
The high abundance of gram-negative compared with gram-positive bacteria could be due to the presence of lysozyme. The mean value of lysozyme concentration of donkey milk used in this study was 2.9 ± 0.9 mg/mL, which is in accordance to literature and is higher than the lysozyme concentration of bovine (0.09 mg/L) and human (200 mg/L) milk (
). The mean value of lysozyme concentration ranges from 1 up to 4 mg/mL among the different donkey breeds. Its hydrolytic activity against the glycosidic bonds of peptidoglycan makes lysozyme more effective against gram-positive bacteria. Indeed, recent 16S rDNA metagenomic studies that performed in other animals' milk with a lower concentration of lysozyme, including goat, sheep, cow, and human, indicated the presence of gram-positive bacteria such as Staphylococcus and Streptococcus, in high percentages (Table 5;
) reported that Staphylococcus and Streptococcus were among the most commonly detected genera between human and cow milk, in addition to Corynebacterium, Pseudomonas, Bacteroides Bifidobacterium, Propionibacterium, and Enterococcus. Apart from lysozyme, a second antimicrobial agent that is present in donkey milk is lactoferrin. Lactoferrin is detected in lower concentration compared with lysozyme (up to 135 μg/mL;
). The capacity of lactoferrin to bind LPS of gram-negative bacteria may provide access to lysozyme's molecules to target and degrade the peptidoglycan in the cell wall. Based on the current findings, though, lactoferrin's presence in limited concentrations might not suffice to prevent the growth of gram-negative bacteria.
The most abundant genus in all donkey milk samples was Sphingomonas. This genus is characterized by the presence of glycosphingolipids instead of LPS in their cell envelopes. Also, they possess the ability to grow in stressful for most bacteria environments (
Identification of soil micro-habitats for growth, death and survival of a bacterium, γ-1,2,3,4,5,6-hexachlorocyclohexane-assimilating Sphingomonas paucimobilis, by fractionation of soil.
). However, spoilage of milk has mostly been attributed to the psychrotrophic members of the genus Pseudomonas. Pseudomonas was detected in increased relative abundances in several milk samples. The capability of Pseudomonas spp. to successfully utilize milk proteins and lipids due to their proteolytic and lipolytic enzymatic activities provides them with the ability to grow in raw milk (
The genus Mesorhizobium comprises soil bacteria that colonize legume roots and assist in the transformation of atmospheric nitrogen into plant-available compounds (
). Forage legumes are important sources of protein, fiber, and energy for animal-based agriculture. Moreover, legume grazing supports meat and milk production, as well as suppressed growth of internal parasites that provoke animals' mortality (
) but in lower relative abundances. Additional nitrogen-fixing bacteria detected, but in limited relative abundances (<1%), include Rhizobium, Azorhizobium, Sinorhizobium/Ensifer, Azospirillum, and Nitrospirillum. The occurrence of these symbiotic bacteria might be associated with donkeys' nutrition, and specifically with legumes (
, working with human milk, suggested that selected bacteria of the maternal gastrointestinal microbiota can access the mammary glands through oromammary and enteromammary pathways. The mechanism involves dendritic cells and CD18+ cells, which would be able to take up nonpathogenic bacteria from the gut epithelia cells and subsequently, to carry them to other locations, including the lactating mammary gland.
The presence of LAB, including Lactobacillus and Streptococcus, was indicated in all donkey milk samples, with average relative abundance of 2.42% (ranging from 1% to 4%). These results are in agreement with the other 16S rRNA gene amplicon-HTS studies of
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
) on donkey milk microbiota, in which the average relative abundances of LAB were 4.2% and 2.55%, respectively. The LAB are commonly detected in milk and dairy samples due to their capacity to ferment lactose successfully (
High throughput sequencing technologies as a new toolbox for deep analysis, characterization and potentially authentication of protection designation of origin cheeses?.
) LAB cocci are more resistant to lysozyme than lactobacilli, and among lactobacilli, the lysozyme sensitivity is species or strain-specific; for instance, thermophilic species are more sensitive than hetero-fermentative mesophilic lactobacilli. The LAB possess antimicrobial properties, mainly due to the presence of bacteriocin-like inhibitory substances (
Application of bacteriocin-producing Enterococcus faecium isolated from donkey milk, in the bio-control of Listeria monocytogenes in fresh whey cheese.
, it was shown that the main LAB isolated from donkey milk samples belong to the Enterococcus species. Most of the isolates had interesting technological properties and were able to produce bacteriocins, whereas no pathogenicity was detected. Their presence in milk restricts the risk of food-borne diseases caused by bacteria, including Staphylococcus aureus, Listeria monocytogenes, pathogenic Escherichia coli, and Salmonella spp. (
In the present study, the application of culture-based approaches identified the presence of staphylococci and Enterobacteriaceae in less than 4.7 and 3.4 log10 cfu/mL, respectively. The 16S rDNA sequencing analysis detected number of reads belonging to the genus Staphylococcus and the family Enterobacteriaceae that corresponded to low relative abundances (0.034% to 0.188% and 0.152% to 2.375%, respectively). Moreover, the culture-based analysis indicated the absence of the species Escherichia coli, Bacillus cereus, and Listeria monocytogenes. Regarding Escherichia coli and Listeria monocytogenes, culture-based results were in agreement with 16S rDNA metagenomic analysis findings. However, some reads corresponding to the genus Escherichia/Shigella were identified (5–52 reads) but assigned to the species Escherichia/Shigella dysenteriae and Escherichia vulneris. Also, HTS identified the presence of Bacillus cereus in 2 species, but in limited relative representation (0.02%). Based on these observations, 16S rRNA gene sequencing analysis showed higher sensitivity to detect specific bacteria than the usual culture-based method.
The present study indicated that donkey milk harbors complex bacterial communities containing different microorganisms. Still, the identification of the origin of the milk microbiota remains largely unknown. In agreement with other studies on donkey milk microbiome (
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
), the plethora of the detected bacteria are considered environmental. Their existence in donkey milk might come from external contamination of the breast (or udder) during nursing, derived from the mother's skin or the infant's oral cavity. This suggestion is supported by observations on human and bovine milk microbiota, in which species found in the teat skin (
) were detected in milk. In addition, some researchers suggest an endogenous origin of the milk microbiome, proposing the presence of a hypothetical enteromammary pathway (
). Interestingly, in the present, as well as other studies on milk microbiota (see Table 5), the detection of LAB, such as Lactobacillus in milk, which contains species associated with the gut microbiome and not detected on the breast skin of humans and animals, supports this theory. Moreover, the presence of alive bacteria in the mammary gland of women who were not breastfed before, indicates also that this tissue might not be sterile (
). Noteworthy, bacteria associated with the animal's nutrition, such as Mesorhizobium, were also found in the present study; their origin though remains unspecified. Overall, more effort needs to be provided to specify whether the rich diversity of microbes detected in donkey milk is shaped due to the contamination of the environment or affected by the animals' nutrition and the microbial communities existing in the animals' gut.
CONCLUSIONS
This is the first study performed to characterize the bacterial communities of donkey milk in a Cyprus farm via HTS. It highlights and confirms that the donkey milk bacterial microbiome is mostly comprised of gram-negative bacteria, possibly due to the increased concentration of lysozyme. In the future, additional donkey milk samples are to be analyzed to enable a broader evaluation and characterization of the existing bacterial communities. Factors that contribute to the conformation of donkey milk microbiota, such as the origin of milk, the environment, animals' health, diet, and genetics, will also be analyzed. The metagenomic analysis could be combined with other methodologies, including proteomics and metabolomics, for a sufficient estimation of the associations among the existing bacteria, the secreted metabolites and the antimicrobial agents detected in donkey milk. These analyses will shed more light on the nutritional benefit and antimicrobial activity of donkey milk.
ACKNOWLEDGMENTS
The authors acknowledge the technical support and welcoming environment from Golden Donkeys Farm, Cyprus, for the milk samples collection. Author contributions: conceptualization, D.T. and P.P.; methodology, E.K., D.A., and M.A.; formal analysis, E.K., M.A., and A.K.; investigation, E.K., D.A. and M.A.; resources, D.T., P.P., and P.M.; data curation, E.K.; writing—original draft preparation, E.K. and M.A.; writing—review and editing, E.K., D.T., and P.P.; supervision, D.T. and P.P.; project administration, D.T.; funding acquisition, D.T. All authors have read and agreed to the published version of the manuscript. Funding: this research was funded by INTERREG Greece–Cyprus 2014-2020 Program [Project AGRO-ID, which is co-funded by the European Union (ERDF) and National Resources of Greece and Cyprus] and project DELIVER by the Research and Innovation Foundation (RIF) of Cyprus. Andreas Kamilaris has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 739578 complemented by the government of the Republic of Cyprus through the Directorate General for European Programmes, Coordination and Development. The authors declare no conflict of interest.
Application of bacteriocin-producing Enterococcus faecium isolated from donkey milk, in the bio-control of Listeria monocytogenes in fresh whey cheese.
Hygienic characteristics and microbiological hazard identification in horse and donkey raw milk (Caratteristiche igienico-sanitarie ed identificazione dei rischi microbiologici nel latte di cavalla e di asina).
ISO 6888-1(1999). Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium..
International Organization for Standardization,
Geneva, Switzerland1999
ISO 16649-2:2001. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli—Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide..
International Organization for Standardization,
Geneva, Switzerland2001
ISO 21528-2:2004. Microbiology of food and animal feeding stuffs—Horizontal methods for the detection and enumeration of Enterobacteriaceae—Part 2: Colony-count method..
International Organization for Standardization,
Geneva, Switzerland2004
ISO 7932:2004. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of presumptive Bacillus cereus—Colony-count technique at 30 degrees C..
International Organization for Standardization,
Geneva, Switzerland2004
ISO 4833-2:2013. Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of microorganisms—Colony-count technique at 30 degrees C by the surface plating technique..
International Organization for Standardization,
Geneva, Switzerland2013
ISO 11290-1:2017. Microbiology of the food chain—Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. Part 1: Detection method..
International Organization for Standardization,
Geneva, Switzerland2017
High throughput sequencing technologies as a new toolbox for deep analysis, characterization and potentially authentication of protection designation of origin cheeses?.
Identification of soil micro-habitats for growth, death and survival of a bacterium, γ-1,2,3,4,5,6-hexachlorocyclohexane-assimilating Sphingomonas paucimobilis, by fractionation of soil.
Microbial populations of fresh and cold stored donkey milk by high-throughput sequencing provide indication for a correct management of this high-value product.
Human milk microbial community structure is relatively stable and related to variations in macronutrient and micronutrient intakes in healthy lactating women.
Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice.
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