What happens when we sequence our cheeses?

It has now been six months since I started my PhD, and I thought it was about time for a progress update! While most of my time at University so far has involved lots of training and reading, I have started on a few experiments and thought I’d talk you through them; the first being DNA sequencing of the cheeses and kefir.

By sequencing the DNA of all the bacteria present in a sample, you can analyse the data to identify what bacteria are there. In January, I sent off eleven samples of cheese and kefir to a company which sequences bacterial DNA to figure out what is in there.

Figure 1 is a relative abundance graph, which shows what different species you have in a sample proportionally. Most of the identified species are bacteria that we add as starter cultures, for example Lactococcus lactis is added to every cheese that we make and is present in all the samples. And to Highmoor and Witheridge we add thermophilic (heat-loving) bacteria Streptococcus thermophilus and Lactobacillus delbrueckii, which you can see in all the Highmoor and Witheridge samples. Glutamicibacter arilaitensis is part of the collection of cultures we add to Highmoor as rind bacteria, and we can see that it’s abundance is increased in the mid- and mature Highmoor as the rind has started growing in comparison to the young Highmoor which had no developed rind.

Another interesting point to highlight is the presence of Propionibacterium freudenreichii, which is the bacteria we add to Highmoor to create the small holes in the middle, which is also added as a starter culture. Similarly Staphylococcus equorum, present in the mature Witheridge is also present in the afore-mentioned collection of cultures which makes up the rind bacteria in Highmoor – it’s presence in Witheridge is likely to be the result of a slight cross-fertilisation which is very possible as Highmoor and Witheridge share the brine baths and space in our ageing rooms. We don’t see much diversity of species in both the Bix samples, this is really just due to the fact that Bix is a yeast and mould dominated cheese, and the method of sequencing used here only detects bacterial DNA. Kefir contains lots of Lactococcus lactis, but also lots of species labelled “other” which I hope to look into further.

So Figure 1 shows us the proportion of each species of bacteria in each sample, and in Table 1, we can see the number of bacterial species identified in each sample regardless of how abundant these species are.

The most interesting take-away from this is the increase in observed species for mid, mature, and raw Witheridge compared to the young Witheridge and all the other samples – the most notable difference here is the addition of hay; the young Witheridge was only a week old when I took the sample, and no hay had yet been added.

These bacteria presumably make up most of the species in the “others” band which makes up a large proportion of the relative abundance graph for these cheeses. Though it is likely that these bacteria weren’t actually alive on the cheese – we sterilise the hay using steam, and the sequencing method detects bacterial DNA regardless of whether it is live or not.

We are interested to look further into this and see if these species of bacteria have any bearing on what makes Witheridge smell, feel, and taste the way it does, as well as considering if the hay has a prebiotic effect on the other microbes present in Witheridge.

To investigate this further we can conduct NMR (nuclear magnetic resonance) analysis on the same samples that were sequenced, and this will tell us what metabolites are present and hopefully we will be able to work out which bacteria are producing which metabolites by looking at the sequencing data. Another interesting thing to point out from these sequencing results is the volume

of species present in kefir – we can now prove that kefir has a very diverse population of bacteria, with over 1100 identified species! To put this into context, Biotiful claims on their website that their kefir contains over 40 strains of bacteria, whilst Yeo Valley kefir contains just 14 species (1,2) .

So there you have it, a hopefully interesting glimpse into what bacterial species are in our cheeses! The next stage of experiments involves looking at how these might effect your gut microbiome in an in vitro model, which I will talk about next time.

References

1. Organic Plain Natural Kefir Yogurt [Internet]. [cited 2023 Jun 2]. Available from:

https://www.yeovalley.co.uk/things-we-make/kefir/natural-kefir

2. Biotiful Dairy Kefir FAQs [Internet]. [cited 2023 Jun 2]. Available from:

https://biotifulguthealth.com/faq/