Human Milk Oligosaccharides (HMOs) and Gut Microbiota, Immune System in Antarctica

The aim of this study is to investigate the effect of supplementation with prebiotics, a specific mixture of carbohydrates found in breast milk (HMOs), as a measure to improve intestinal flora, inflammatory processes in the gut, general immune status, carbohydrate regulation, fat and bone metabolism and neurological changes in Antarctica. The approximately one-year stay at Concordia Station (Antarctica) at an altitude of about 3200 m includes a long journey and subsequent extreme environmental conditions as well as corresponding adaptation mechanisms and is therefore a very good terrestrial model (analogue model) for investigating changes during space stays. Long-term (i.e. > 6 months) exposure to microgravity leads to numerous physiological responses, including impaired glucose tolerance, reduced immune function and bone loss, which can lead to quite severe health consequences. In this analogue model, similar changes, albeit less severe, are observed with regard to the immune system or glucose tolerance as in space flight.

The composition of the diet significantly influences the composition of the intestinal flora. In particular, dietary carbohydrates provide the substrate for certain bacteria that could have a positive effect on health. Prebiotics are defined as substrates that are selectively used by host microorganisms and that provide health benefits. Prebiotics are digested only by gut microorganisms and stimulate the growth or activity of bacteria in the gut that have shown health benefits or improved well-being. The main nutrients for bacterial growth are non-digestible carbohydrates, which by definition are not digested by the host, in this case the human organism (host). Non-digestible carbohydrates include, for example, resistant starch and dextrins, non-starch polysaccharides (NSPs) (e.g. pectins) and non-digestible oligosaccharides (e.g. Raffinose, inulin, etc.). These carbohydrates are fermented by the intestinal bacteria to short-chain fatty acids (SCFAs), mainly acetic acid, propionic acid and butyric acid. This process takes place mainly in the large intestine, but also in the small intestine. SCFAs are rapidly absorbed by the mucosa of the large intestine and exert whole-body effects, contributing, for example, to the host's energy needs or mediating gut-brain communication, which has a major impact on the physiology and health of the host.

HMOs are also prebiotics and are the third most abundant solid component in breast milk. Most of the research published to date on the effects of HMOs has therefore been conducted on infants. HMOs are not digested in the intestine and are mainly utilised in the large intestine by the bacteria that reside there. In infants, it has been shown that HMOs not only promote a healthy composition of the intestinal flora, but also impair the growth of pathogens and modulate the expression of genes involved in inflammation. Several in vitro and in vivo studies have shown antiviral properties of HMOs against various viruses such as rotavirus, norovirus, HIV and influenza. HMOs play a significant role in the immune system by enhancing the immune response. Initial applications of HMOs in adults have supported these findings.

Therefore, this study aims to investigate how daily administration of 5.5 g of an HMO mixture during hibernation at Concordia Station in Antarctica affects:

• glucose tolerance
• inflammatory processes
• immune function
• fat and bone metabolism and
• Well-being

Study procedure This is a so-called placebo-controlled, randomised intervention study in parallel design. This means that subjects will be randomly selected to receive either the mixture of HMOs daily or a placebo. The study duration for a test person is approx. 20 months. It is divided into an examination phase before departure to Antarctica, approximately in September of a year, six phases during the wintering in Antarctica and one phase approximately 6 months after return.

Each individual phase is identical, one example shown in Figure 1.

Day 1 (Weekend day) Day 2 Day 3 Day 4 Day 5 Fasting blood collection X Oral glucose tolerance-test X Saliva sample X Feces sample X X X (X) Diet log X X X HMO supplementation Daily, onset after first session at Concordia

Figure 1: Representation of each phase (U as in figure 1) of testing

Intervention The intervention, i.e. the treatment measure used in this study, in one group is the daily administration of 5.5 g of a carbohydrate mixture analogous to human milk but produced by bacteria. This amount is certified by the European Control Authority as safe for human consumption. The other group receives a corresponding placebo, Maltose.

Measurement method Blood test and venous access Blood sampling from the peripheral vein is associated with the usual risks of venipuncture. These are haematomas or infections at the puncture site. When a peripheral venous access is placed, in rare cases there may be injuries to the vein (sclerotherapy) and the peripheric nerves. Blood collection and venous access are performed by an experienced doctor, which minimises the risk.

Fasting blood collections Fasting blood samples for analysis of various blood parameters are taken on the same day as the oral glucose tolerance test (OGTT) (see figure 1). Since several blood samples are taken for the OGTT, also after the drink has been taken, a venous access in the form of an indwelling venous cannula (small plastic tube) will be placed in the area of the forearm in order to avoid repeated puncturing of the arm veins.

Glucose tolerance by means of an oral glucose tolerance test The so-called oral glucose tolerance test (OGTT) can be used to determine differences in glucose metabolism (sugar metabolism) between the intervention and control phases. On approximately day 3 of each study phase, a drink will be consumed in the morning, fasting state, which is prepared from 75 g glucose (Medicalfox Gluko 75 powder) and 300 ml drinking water. Blood is taken every 15 minutes in the first hour after the drink is given, and every 30 minutes in the second hour.

Saliva sample Saliva samples will be taken to test certain hormones that reflect stress levels, as well as the reactivity of certain viruses (herpes virus, rotavirus). For this purpose, a small cotton wool roll from a prepared container (salivette) is chewed in the mouth for several minutes and returned to the collection tube once during the examination phase. The saliva obtained is frozen and later analysed in the laboratory.

Faecal collection In order to examine the changes in the intestinal flora, faeces samples are taken on 3 consecutive days in each phase. For this purpose, a so-called 'Feces Catcher' is placed over the toilet to better collect the faeces. Three samples are then taken from these faeces in 3 different tubes. The small spoon in each tube is filled with faeces, put back into the tube, closed and frozen.

Documentation of the diet In order to be able to estimate the influence of the food composition on the intestinal flora, the food consumed is documented on 2 working days and one weekend day. This is done in a prepared dietlog.

Inclusion criteria

• Physically and mentally healthy subjects
• Volunteers that are able and declare their willingness to participate in the entire study
• Fasting blood glucose concentration: <120 mg/dL
• Willing to be assigned randomly either to the treatment or the control group
• Successfully pass the medical screening
• Signed informed consent
• Social insurance

Exclusion criteria

• Medication that may interfere with the interpretation of the results
• Recent sub-standard nutritional status
• Abuse of drugs, medicine or alcohol
• Participation in another study up to two months before study onset
• Cannot clear a criminal background check
• No signed consent form before the onset of the experiment
• Blood donors in the past three months before the onset of the experiment
• Vegetarian and Vegans