Key words
Microbiota, biodiversity, fermentation, cow’s milk, amasi, allergy
prevention, rural.
To the Editor,
The anti-allergic properties of raw and fermented cow’s milk are being
explored globally.1,2
Traditionally fermented milk (isiXhosa - ‘amasi ’) and unfermented
(unpasteurized) cow’s milk (isiXhosa - ‘ubisi ’), are consumed
regularly by rural South African communities. To produce amasi ,
unpasteurized milk is left for three to five days to naturally ferment
at room temperature. The South African Food Sensitisation and Food
Allergy (SAFFA) study, demonstrated that children living in an urban
environment had significantly higher rates of allergic diseases compared
to their rural counterparts, and the consumption of fermented milk was
associated with lower rates of allergic rhinitis, atopic dermatitis and
self-reported asthma.3 Lactic acid producing bacteria
produce important by-products and end-products during milk fermentation
which may contribute to their anti-inflammatory and anti-allergic
properties.4 In this study we characterize and compare
the bacterial microbiota in raw cow’s milk (collected from urban and
rural farms), and home-made and commercially fermented milk products by
high throughput 16S rRNA gene amplicon sequencing. This study received
ethical approval from the University of Cape Town (animal ethics:
018_033).
Rural fresh cow’s milk samples were collected from three farms in rural
South Africa. Urban samples were collected from a farm in Cape Town.
Before milking commenced, the cows and the udders were declared in a
“heathy state” by each farmer. The udders were not cleaned or washed
prior to milking, as these procedures were not included in the normal
milking routine on the farms. Samples were labelled as Rural fresh
cow’s milk 2 , Rural fresh cow’s milk 3, Urban fresh cow’s milk
1 , Urban fresh cow’s milk 2 and Urban fresh cow’s milk 3and transported frozen to the laboratory. A further sample of
unpasteurized milk obtained from a separate farm in the same rural area
labelled as Home fermented milk (amasi) , was sealed, and left for
five days at ambient temperatures to allow natural fermentation. Three
different brands of commercially fermented amasi were obtained
and labelled as Comm-othando , Comm-amyoli andComm-maas . All samples were analyzed by the Centre for Proteomic
and Genomic Research (CPRG), Cape Town, South Africa.
DNA was extracted from the milk samples using the ZymoBIOMICS® DNA
Miniprep Kit (Zymo Research, USA).
The V3-V4 variable region of the 16S rRNA gene was amplified from 2.5ng
to 25ng of purified DNA by 25 cycles of PCR and barcoded for
multiplexing using the Nextera® XT Index kit (Illumina, USA) and KAPA
HiFi DNA Polymerase (Roche®, USA). The nine milk product samples, a
positive control (ZymoBIOMICS® Microbial Community DNA standard) and a
negative control (DNA suspension buffer) were included in library
preparation. The size of the libraries was verified using an Agilent®
2100 Bioanalyzer (Agilent, USA). Library concentration was evaluated
using the KAPA Illumina Library Quantification Kit (Roche). The
libraries were sequenced on an Illumina MiSeq sequencer at the CPRG
using a MiSeq Reagent Kit v2 (Illumina®) to produce paired-end 250 base
pair reads.
Illumina MiSeq read quality
assessment and taxonomic profiling were performed on a high-performance
compute cluster using a custom Nextflow pipeline
[https://github.com/h3abionet/TADA], implementing
FastQC5 and MultiQC6 for quality
control, dada27 for ASV prediction, and the RefSeq-RDP
16S database (v3 May 2018) for taxonomic annotation.8All downstream analyses were performed in R, with custom functions
[https://gist.github.com/kviljoen/97d36c689c5c9b9c39939c7a100720b9].Taxa
(merged at the lowest available taxonomic level, tax_glom.kv function)
were deemed significantly different (in terms of abundance and/or
absence/presence) between fermented versus unfermented samples if they
exhibited a fold change (beta coefficient) of ≥ 1.5 and had an adjusted
p-value of ≤ 0.05 and if at least one of the two groups compared had ≥
60% of samples with the given ASV/taxon, or, if the result of Fisher’s
exact test was significant (after multiple-testing correction by the
Benjamini-Hochberg method), using the R package metagenomeSeq, and
custom function super.fitZig.kv.
Results showed that the fresh cow’s milk samples (both urban and rural)
had significantly higher numbers of ASVs and identified taxa compared toamasi and commercially fermented milk. All three commercially
fermented samples appeared similar and compared to amasi , had
lower numbers of ASVs and merged taxa. The commercially fermented milk
had the lowest Simpson alpha diversities. The Shannon alpha diversity
was high in the three urban fresh cow’s milk samples. The commercially
fermented milk samples were very similar and low in diversity. The
Shannon alpha diversity of the two rural fresh cow’s milk samples, was
markedly dissimilar (Figure 1). Principal coordinates analysis, based on
Bray-Curtis distances, was used to examine the dissimilarities between
different cow’s milk samples’ microbiota communities. The four
differently sourced milk groups (rural fresh, urban fresh, amasi ,
and commercially fermented) were strikingly dissimilar. The home
fermented milk sample amasi and the two rural fresh milk samples,
were uniquely dissimilar from each other and from all the other milk
samples (Figure 2).
Commercially fermented products were similarly dominated by
lactic acid producing bacteria,
belonging to the phylum Firmicutes (more than 98% abundance) and
the phylum Proteobacteria (less than 2% abundance). Theamasi sample comprised approximately 50% Firmicutes and
approximately 50%Proteobacteria . Rural fresh
milk 1 was comprised almost completely of Proteobacteria with
small percentages of Bacteroidetes , Firmicutes andCandidatus Saccharibacteria (Figure S1).
Commercially fermented products appeared remarkably similar at genus
level, with very low richness, comprising of mainly two genera:Lactococcus (more than 75% relative abundance) andLeuconostococcus (about 24% relative abundance). Theamasi sample appeared to have higher richness than commercially
fermented milk, but less than the fresh milk samples. In amasi ,Lactococcus had the highest relative abundance, but the genusLeuconostococcus was absent. Furthermore, in amasi , the
genera Kluyvera , Citrobacter , Streptococcus ,Lactobacillus and Salmonella were present (Figure S2).
Although lactic acid producing bacteria were identified in the fermented
milk products, the presence of these organism in the fresh milk samples,
were inconsistent. In amasi and in the commercially fermented
products, Lactococcus lactis was the most abundant organism.Lactobacillus paracasei was abundant in amasi , but almost
completely absent in all the other milk samples (Figure 3).
Microbiota composition was compared in fermented (home and commercially)
versus unfermented milk. Because of the relatively small number of
samples used in this study, all fermented milk samples were included for
statistical comparison against the unfermented milk samples.
Results for differential abundance testing clearly show significant
difference in Lactococcus and Leuconostococcus species
between the fermented and unfermented milk groups. There are several
taxa that uniformly dominated in commercially fermented milk,
contributing to its evenness and decreased diversity. Lactococcus
lactis dominated in the fermented samples, including amasi .Lactococcus chungangensis had similar abundance across
commercially fermented products, was absent in amasi and present
at exceptionally low levels in the fresh cow’s milk products.Leuconostococcus mesenteroides and Leuconostococcus
pseudomesenteroides were absent in amasi . Lactobacillus
paracasei was abundant in amasi , with low to no occurrence in
all the other samples (Figure S3).
Potential milk pathogens were also differentially abundant between
fermented and unfermented milk. Salmonella enterica in addition
to being present in all fresh cow’s milk samples, was also abundant inamasi , but absent in commercially fermented products.Escherichia/Shigella was present in urban fresh samples and
absent in rural and fermented milk
samples
(Figure 3; Figure S3).
In our study, the genus Lactococcus uniformly dominated in the
commercially fermented milk samples and was also abundant in theamasi sample. An important aim of our study was to compare the
microbiota (at species level) of fermented versus unfermented milk
products, to indicate whether fermentation (either commercially or home
fermented) had significant influences on the occurrence of certain taxa.Lactococcus lactis in the fermented group, reached the highest
statistical difference between the two groups. Lactococcus
chungangensis , Leuconostococcus mesenteroides ,Leuconostococcus pseudomesenteroides and Lactobacillus
paracasei were also significantly more abundant in the fermented milk
group. Potential human pathogens were identified in fresh cow’s milk andamasi . Noteworthy, these organisms were absent in the
commercially fermented products.
Our study was of small numbers and limited to bacteria without assessing
viral, parasitic, or fungal differences. Concerns about contaminant DNA
(e.g. from the laboratory environment, laboratory technicians and
nucleic extraction kits) and cross contamination (e.g. DNA from other
laboratory samples and sample runs) when analyzing low microbial biomass
samples, have been published.9
The consumption of fermented milk appears protective in our setting.
Traditional home fermentation is time consuming and supplanted by the
ingestion of commercially fermented milk. Although low in diversity,
commercially fermented milk appears safe, accessible and its microbiota
composition appears to be consistently abundant of lactic acid producing
bacteria. It is therefore not surprising that commercially soldamasi and yogurt are advised to children from one year of age in
the recently updated South African Food-Based Dietary
guidelines.10 The consumption of commercially
fermented milk should be encouraged in urbanized, modern population
groups, with a high prevalence of allergic disease.