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How do we investigate changes to the gut microbiome?

Now that we have an idea of what bacteria are in the cheeses, we can start to have a look at what the cheese does in the gut. This will eventually involve a full gut model with different modules representing different parts of the colon, but for now I am looking at a smaller model which represents the distal part of the colon where most bacterial activity takes place.

Firstly, we need to need to conduct a simulation of upper gut digestion on the cheese as putting cheese directly into this model would not be representative of what happens in the body, as digestion in the upper gut has already taken place. This procedure involves simulation of chewing while adding the enzymes present in saliva, followed by adding the enzymes present in the stomach, and then those present in the smaller intestine, all while incubating at human body temperature in a shaking incubator.

Overnight the solution then sits in salt water within permeable tubes which represents the way some of the digestion products will leave the gut and enter the blood stream. After this the samples are all freeze dried so they are in a state that we can add to our models.

Figure 2 below

Figure 2: Two different steps of predigestion. A: the cheeses in bottles prior to the small intestine stage of digestion, here I was adding the next set of enzymes (including bile and pancreatin) and adjusting the pH of the solution to represent that of the small intestine. B: the cheeses in their dialysis tubes having incubated in salt water overnight. This is what the cheeses look like when they reach your distal colon!

Now that we have our partially digested cheeses, we can add them to our distal colon models. These are essentially little glass jars with lots of ports for taking samples, measuring and adjusting the pH, adding nitrogen gas to make the jar anaerobic (without oxygen, as it is in the gut), and a jacket around the outside through which water flows to keep the mixture at body temperature.

We add media to this jar containing different salts, enzymes, and vitamins to represent what is naturally present in the gut. Then we can add our digested cheese, followed by some gut bacteria from a faecal sample from a healthy person. This experiment then runs for the next 48 hours, and at certain intervals we take samples of the media which will then be analysed for bacterial content.

In figure 3 you can see how our batch culture is set up, in these initial batch cultures I have been running I have tested all three mature cheeses plus the raw Witheridge. I do this experiment three times with different donors for the faecal sample; repetition is really important in science so that you know any consistent results you get are reliable and not just a fluke. I will then scale up the experiment, having got the hang of how to do it, with cheeses of different stages of maturing and also kefir.

Figure 3 below

Figure 3:

A: One of my five vessels; 1: the gas filters and pipes feeding the media with N2 to keep it anaerobic, the one on the left lets the gas in, the one on the right lets the gas out. 2: this little stopper keeps the sample port sealed, and through this port we put our media, the cheese, and the bacteria into the vessel. 3: this is a pH feeder linked to the box you can see behind the vessel, it pumps either acid or alkali into the vessel to keep it at the pH that is naturally in the gut, around 6.8. 4: just behind the pH feeder you can just about see the top of the pH probe, this is also connected to the box and continually measures the pH inside the vessel. 5: these tubes connect all the vessels to each other and connect back to a water bath running at 37°C which pumps water through all the vessel jackets to keep the media warm. 6: the brown liquid inside the vessel is our media with cheese and microbes added. 7: this plinth is a magnetic stirrer, a magnet inside spins which causes a small magnetic flea inside the vessel to also spin which keeps the media stirring.

B: The full set up; I ran five vessels in this batch culture, from left to right we have the control vessel which had no cheese added, the Bix vessel, the Highmoor vessel, the Witheridge vessel, and the raw Witheridge vessel.

So there you have it, all the experiments I have done so far. The next steps will be to do the analysis of my samples taken from the batch cultures in a process called FISH – fluorescence in situ hybridisation! This attaches little fluorescent probes to the DNA of different bacteria, so we can identify which groups of bacteria are thriving in an environment with cheese in it. I am also very shortly going to do some NMR (nuclear magnetic resonance) studies on the cheeses which will show us what metabolites are in the cheeses, which we can hopefully link back to the sequencing study and we can work out which bacteria are producing which metabolites and therefore how this might relate to how we experience the cheese when we eat it. I’ll give you another update in six months, and I’ll see you next month with a dive into fermented foods!

Sabrina Longley Nettlebed Creamery Microbiologist and Cheesemaker

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