Comparative analyses of the bacterial communities present in the spontaneously fermented milk products of Northeast India and West Africa

Abstract

Introduction: Spontaneous fermentation of raw cow milk without backslopping is in practice worldwide as part of the traditional food culture, including "Doi" preparation in earthen pots in Northeast India, "Kindouri" of Niger and "Fanire" of Benin prepared in calabash vessels in West Africa. Very few reports are available about the differences in bacterial communities that evolved during the spontaneous mesophilic fermentation of cow milk in diverse geographical regions.

Methods: In this study, we used high throughput amplicon sequencing of bacterial 16S rRNA gene to investigate 44 samples of naturally fermented homemade milk products and compared the bacterial community structure of these foods, which are widely consumed in Northeast India and Western Africa.

Results and discussion: The spontaneous milk fermentation shared the lactic acid bacteria, mainly belonging to Lactobacillaceae (Lactobacillus) and Streptococcaceae (Lactococcus) in these two geographically isolated regions. Indian samples showed a high bacterial diversity with the predominance of Acetobacteraceae (Gluconobacter and Acetobacter) and Leuconostoc, whereas Staphylococcaceae (Macrococcus) was abundant in the West African samples. However, the Wagashi cheese of Benin, prepared by curdling the milk with proteolytic leaf extract of Calotrophis procera followed by natural fermentation, contained Streptococcaceae (Streptococcus spp.) as the dominant bacteria. Our analysis also detected several potential pathogens, like Streptococcus infantarius an emerging infectious foodborne pathogen in Wagashi samples, an uncultured bacterium of Enterobacteriaceae in Kindouri and Fanire samples, and Clostridium spp. in the Doi samples of Northeast India. These findings will allow us to develop strategies to address the safety issues related to spontaneous milk fermentation and implement technological interventions for controlled milk fermentation by designing starter culture consortiums for the sustainable production of uniform quality products with desirable functional and organoleptic properties.

Keywords: Acetobacter; Gluconobacter; Lactobacillus; Lactococcus; Macrococcus caseolyticus; MiSeq amplicon sequencing; Streptococcus infantarius; spontaneously fermented milk products.

Figure 1

The bacterial community compositional difference in the spontaneously fermented milk products of Northeast India and West Africa. The spontaneously fermented milk products “Doi” in the traditional earthen pot (A) in Northeast India and “Fanire” in the traditional calabash vessel (B) in the Fulani camp of Benin are shown here. The taxon bar chart shows the family-level (C) and genus-level (D) differences in the relative abundance (%) of predominant bacteria present in the fermented milk products of Northeast India and West Africa. The sample details are available in Table 1.

Figure 2

Differences in bacterial diversity in the spontaneously fermented milk products of Northeast India and West Africa. (A) PCoA biplot based on Bray-Curtis dissimilarity of the species-level OTUs shows a significant difference in the overall bacterial community structure between the spontaneously fermented milk products of the two continents. The significance of the difference is expressed as a Bonferroni-corrected p-value (q = 0.0001, F = 12.0, PERMANOVA). (B, C) The boxplot shows higher bacterial diversity in Indian fermented milk products (Chao species richness and Shannon diversity index) than in West African samples. The significance of the difference was calculated using Student's t-test and indicated as ***p<0.0001.

Figure 3

A hierarchically clustered heat map shows the significantly differing bacterial genera between the spontaneously fermented milk samples of Northeast India and West Africa. The bacterial genus with a relative abundance of more than 1% and significantly different between the two continents (Wilcoxon test, q < 0.001, BH corrected) is listed here. The abundance difference is shown as a red and blue colour gradient key.

Figure 4

The difference in the overall bacterial community structure in the uncoloured (A) and coloured Wagashi cheese (B) marketed in Benin. The taxon bar chart shows the difference in the relative abundance (%) of predominant bacteria present in the uncoloured Wagashi cheese and coloured Wagashi cheese at the family level (C) and genera level (D).