Longevity and the microbiome

A targeted combination of beneficial bacteria Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, pomegranate extract, polyphenols, and vitamins B2 and D3.

The combination of probiotic bacteria Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, pomegranate extract, polyphenols, and vitamins B2 and D3 was chosen on the basis of their scientifically demonstrated positive anti-inflammatory properties, effects supporting healthier and slower ageing, and ability to protect against diseases of civilisation.

I. Clinical study

• 6-month
• randomised
• double-blind
• placebo-controlled
• in a group of 97 older adults

Consumption of the strains L. rhamnosus IMC 501 and L. paracasei IMC 502 has a favourable anti-inflammatory effect, through modulation of the gut microbiota and a subsequent increase in the production of SCFAs (short-chain fatty acids). These strains also slow the age-related degradation of the gut microbiota, thereby helping to reduce inflammation. Including them in the daily diet of older adults may therefore represent a preventive strategy to support healthy ageing.

Inflammation, ageing and the gut microbiome

The global population is ageing faster than ever before (1), with life expectancy at birth reaching 72.8 years in 2019, an increase of almost 9 years compared with 1990 (2). Maintaining an active lifestyle in old age will be key in the future, as more than 50% of adult morbidity worldwide is attributed to age-related diseases (3).

Ageing is associated with chronic, low-grade inflammation, which is a risk factor for the development of several diseases. Ageing markedly affects intestinal homeostasis and dietary and lifestyle habits and, together with physiological age-related changes, causes dysbiosis of the gut microbiota in older adults. This dysbiosis, together with low-grade inflammation, may contribute to the development of diseases and to the increasing frailty of older people.

To evaluate the influence of a balanced gut microbiota in suppressing the inflammatory processes associated with ageing, a randomised, double-blind, placebo-controlled study was carried out, aimed at assessing the effect of an intervention with a combination of the probiotic strains L. rhamnosus IMC 501 and L. paracasei IMC 502 on healthy ageing.

Methods

The study evaluated the effect of daily consumption of a probiotic mixture containing 5x10⁹ CFU of L. rhamnosus IMC 501 and L. paracasei IMC 502 in a 1:1 ratio, given in the form of fortified foods or a food supplement, in healthy older adults.

The aim of the supplementation was to improve parameters related to ageing, inflammation and the participants’ overall wellbeing. Participants were randomly assigned to one of two parallel groups: one group received probiotic-fortified foods and capsules, the other group received matching placebos. The primary endpoint of the study was the plasma concentration of hsCRP (high-sensitivity C-reactive protein). Secondary endpoints included monitoring selected groups of gut microbiota bacteria and levels of SCFAs (short-chain fatty acids) in stool samples.

Results

hsCRP level

CRP plays an important role in inflammatory and disease processes and in the host’s responses to infection. Its normal levels rise with ageing. Raised serum levels of it have been found to accompany increased susceptibility to disease and mortality in older patients, such as an increased risk of sarcopenia, cardiovascular pathologies, disability and cognitive decline in individuals over 65 (4).

To compare hsCRP levels between the placebo and probiotic groups, the difference between the values before (T0) and after (T1) supplementation was used.

Although the number of participants in the study was not high, the fall in hsCRP levels was statistically significantly greater in the probiotic group than with placebo (p < 0.05), which suggests that probiotic supplementation had a positive effect on chronic low-grade inflammation. When different durations of probiotic administration were compared, it turned out that six months of supplementation had a more significant effect on lowering the hsCRP level than a shorter duration (p < 0.05) (Fig. 1).

Monitoring of selected bacterial groups

The gut microbiota profile was examined before and after probiotic administration in order to monitor any changes that occurred, taking into account the mean values of the placebo and probiotic groups. No significant changes were recorded in the placebo group apart from a statistically significant fall in bifidobacteria at T1. The main finding in the probiotic group was a significant increase in Lactobacillus spp. In addition, in the probiotic group, long-term administration had a positive effect on the efficacy of the supplementation itself, leading to a significant improvement in Lactobacillus spp. counts in 100% of the subjects who continued taking it for 6 months, compared with the group that took supplements for less than 6 months. The proportion of people with increased/decreased bifidobacteria concentrations after the intervention was also observed. In particular, Bifidobacterium spp. increased in 76% of participants in the probiotic group, with a mean ratio of 6.3-fold. The opposite trend, with falling values, was observed in 63% of participants in the placebo group.

Gut microbiota analysis

To characterise the effects of probiotic supplementation on the participants’ gut microbiota, the V3–V4 regions of the 16S rDNA genes were sequenced using the Illumina MiSeq sequencing platform. The composition of the gut microbiota in the probiotic group was analysed at two time points: before the intervention (T0) and after it (T1).

Composition of the microbiota at the level of phyla

A comparison before and after the intervention showed a significant reduction in Proteobacteria, which are associated with inflammation and gut dysbiosis, and a significant increase in Verrucomicrobia and Actinobacteria, which suggests positive modulation of the microbiota.

Changes at the level of genera and families

Within Verrucomicrobia, a marked increase in Akkermansiaceae (family) and Akkermansia spp. (genus) was observed. Within Actinobacteria, the abundance of Bifidobacteriaceae (family) and Bifidobacterium spp. (genus) increased. By predicting the functional profile of the microbiota (using the PICRUSt tool), the metabolic pathways present in the gut microbiota were estimated on the basis of 16S sequences. After probiotic supplementation, a significant increase in the activity of several metabolic pathways was shown, especially in the area of the breakdown of amines, aromatic compounds and amino acids.

Characterisation of the SCFA (short-chain fatty acid) profile

At the start of the study, large individual variability in SCFA levels was recorded between participants, as well as between different groups and sexes. After supplementation ended, the probiotic group was found to have a marked, although statistically non-significant, increase in total SCFA levels compared with placebo. When values before and after the intervention (T0 vs T1) within the same group were compared, a statistically significant increase in total SCFAs and butyric acid (butyrate) was found in the probiotic group. Acetic acid (acetate) was the most abundant SCFA in both groups. After the intervention, a slight but statistically non-significant increase in its level was recorded in the probiotic group. Levels of the other SCFAs remained relatively unchanged, with no statistically significant differences. This result suggests that long-term consumption of probiotics may support the production of beneficial SCFAs, especially butyrate, which plays a key role in protecting the intestinal barrier and reducing inflammation.

II. Clinical study

• 6-month
• randomised
• double-blind
• placebo-controlled
• in a group of 97 older adults

Long-term supplementation with the strains L. rhamnosus IMC 501 and L. paracasei IMC 502 may contribute to maintaining nutritional status, physical performance and muscle function in healthy older adults. The results suggest that modulation of the gut microbiota may be an effective preventive strategy against sarcopenia.

Methods

The aim of this study was to evaluate the modulating effects of probiotic supplementation on inflammatory markers and the nutritional status of older adults. Participants were randomly assigned to one of two groups. The intervention group received a mixture of the strains Lacticaseibacillus rhamnosus IMC 501 and Lacticaseibacillus paracasei IMC 502 in a 1:1 ratio, at a dose of 5 × 10⁹ CFU per day. The placebo group received an identical product without probiotics. Before (T0) and after the 6-month intervention (T1), stool, urine and blood samples were collected to determine serum cytokines, biogenic amines and inflammatory markers. In addition, the Mini Nutritional Assessment (MNA) questionnaire was completed to record nutritional status.

Results

Detection and quantification of biogenic amines

Biogenic amines (BAs) have recently been identified as diagnostic markers of various and serious diseases (5). The method used made it possible to quantify 11 biogenic amines (BAs) in each urine sample. At time points T0 and T1, the results showed large between- and within-group variability among participants, which is expected with a biological matrix such as urine. The total amount of BAs fell after the probiotic intervention, whereas it rose in the placebo group. In both cases, however, the changes were not statistically significant. In general, apart from 2-phenylethylamine, which rose in the probiotic group at T1, we observed a falling trend in urinary BA concentration in the probiotic group at the end of supplementation. A significant fall was recorded for histamine. The dopamine level fell in the probiotic group, while serotonin levels remained almost stable.

Evaluation of TNF-α and IGF-1

TNF-α (tumour necrosis factor alpha) is a pro-inflammatory cytokine, i.e. a molecule produced by the immune system that promotes inflammation. Its level rises with age, which contributes to the so-called chronic low-grade inflammation associated with ageing (6).

In serum, TNF-α levels were higher than the normal reference values (<20 pg/mL), with means exceeding 50 pg/mL. After supplementation there was a significant fall in TNF-α levels in the probiotic group.

IGF-1 (insulin-like growth factor 1) is an insulin-like growth factor that plays a key role in the growth and regeneration of tissues, especially muscle. Its production falls naturally with age, which contributes to sarcopenia (loss of muscle mass and strength) (7). The IGF-1 level in the probiotic group at T1 rose significantly compared with both baseline and the placebo group at T1 (Fig. 2).

Detection and quantification of cytokines

The cytokine profile was assessed in serum as well as in stool. In serum, it was assessed before and after supplementation in both groups. Most cytokines showed no significant changes after the intervention, with the exception of IL-6, IL-8 and MIP-1α, which fell significantly in the probiotic group after supplementation.

The stool cytokine results showed that the probiotic intervention did not change the stool cytokine profile in either group.

Assessment of nutritional status

After the 6-month intervention, the probiotic group showed a statistically significant improvement in the risk of malnutrition: the percentage of people at risk of malnutrition fell from 54.8% to 35.7%, and the percentage of people with a normal nutritional status rose from 28.6% to 57.1%. The number of malnourished people also fell.

Conclusion

The results of the studies show that long-term supplementation with a probiotic blend of Lacticaseibacillus rhamnosus IMC 501 and Lacticaseibacillus paracasei IMC 502 can alleviate inflammatory processes associated with ageing by modulating inflammatory and nutritional parameters.

Polyphenol protection against the ageing of the body

Although the WHO recommends eating at least five portions of fruit and vegetables a day, only a small part of the population follows this recommendation (8). In Europe, only around 12% of the population eat the recommended amount daily (9). A low intake of plant-based food is associated with a higher incidence of chronic diseases, inflammatory processes and increased mortality (10). The consequence is also accelerated biological ageing processes, which are closely linked to oxidative stress and disorders of vascular homeostasis (11).

Polyphenols are a group of bioactive compounds naturally present in plant foods (12). They are among the most important antioxidants in human nutrition and play a key role in the prevention of oxidative stress, inflammatory processes, neurodegenerative and cardiometabolic diseases (13). Their biological activity depends on a regular and varied intake, as they are not stored in the body and are excreted quickly (14).

Clinical and observational studies show that a higher intake of polyphenols is associated with lower levels of inflammatory biomarkers, a lower rate of lipid peroxidation, e.g. a fall in malondialdehyde (MDA), and improved endothelial function (the ability of the endothelium to regulate the activity of blood vessels), which may have a significant influence on processes related to longevity and the preservation of functional vitality (15).

A standardised fruit and vegetable complex containing more than 100 polyphenol compounds in a single dose provides a physiologically relevant 24-hour phenolic load, mimicking the effect of a varied plant-based diet over the course of the day. Its absorption kinetics show a three-phase profile. This course ensures a continuous circulation of phenolic metabolites, which leads to even antioxidant protection throughout the day and contributes to maintaining metabolic homeostasis. Clinical data from a double-blind, randomised, placebo-controlled study further demonstrated that after a single dose of the extract there was a significant reduction in MDA levels – a precise biomarker of lipid peroxidation, i.e. damage to fats – in the postprandial period, that is, the period after eating (16). This period is critical for metabolic health, because after meals rich in fats and carbohydrates the production of reactive oxygen species (ROS) increases, which can trigger oxidative stress and promote inflammatory processes (Fig. 3).

The efficacy of polyphenols in this context points to their potential in protecting against acute metabolic fluctuations, which are an important risk factor for cardiovascular and degenerative diseases associated with ageing.

At the same time, an inverse correlation was observed between the concentration of circulating phenolic metabolites and the 24-hour variability of mean arterial pressure (MAP), which suggests a positive influence on vascular regulation. Blood pressure stability throughout the day is an important marker of a healthy vascular system and one of the predictors of longevity (17).

These findings support the use of complex plant polyphenols as part of a nutritional strategy aimed at healthy ageing and the prevention of chronic diseases.

Urolithins and longevity

Plant sources such as pomegranate (Punica granatum), walnuts and raspberries (Rubus idaeus) are a rich source of ellagitannins – polyphenolic compounds with a demonstrated biological effect (18). After oral intake, ellagitannins are hydrolysed in the gastrointestinal tract to ellagic acid (EA), which is then metabolised by the gut microbiota into bioactive metabolites known as urolithins, mainly urolithin A (UA), urolithin B (UB) and isourolithin A (IsoUro-A). Urolithins, especially UA, have a potential positive influence on the ageing process, primarily by supporting cell health. UA can improve mitochondrial function by stimulating gene expression (it switches on or boosts the activity of certain genes that control the repair and performance of mitochondria) and by increasing autophagy – that is, the natural process of cell renewal, which declines with age. In this way it contributes to more efficient removal of damaged cell parts and supports cell regeneration (19).

In addition, an anti-inflammatory effect was observed with UA, namely a reduction in the concentrations of inflammatory biomarkers such as CRP, IL-1β and TNF-α. Since chronic inflammation is one of the key characteristics of ageing, modulating these parameters has high clinical significance. UA also showed a positive influence on muscle function, including improved muscle strength and endurance, especially in the lower limbs. These effects have the potential to contribute to preventing or slowing the progression of sarcopenia, a pathological condition associated with loss of muscle mass and impaired functional capacity in older people (20).

The pharmacokinetic and metabolic properties of a pomegranate extract standardised to its content of ellagitannins, specifically punicalagins, were investigated in two clinical studies. In 2023, a randomised study was carried out to examine the metabolism of the main polyphenol punicalagin and its gut metabolites – UA and UB. Subjects received a single dose of 250 mg or 1000 mg of the extract, with plasma samples taken over 48 hours (21). Another open-label study in humans investigated the absorption, metabolism and antioxidant effect of the extract (22).

Results

Absorption and antioxidant effect of punicalagin

The maximum plasma concentration (Cmax) was 33 ng/ml, 1 hour (Tmax) after administration. Metabolites such as urolithin A, urolithin B, hydroxy-urolithin A, urolithin A glucuronide and EA dimethyl glucuronide were then identified in the plasma using HPLC-MS.

In addition, as early as 30 minutes after consuming the extract, a 32% increase in the antioxidant capacity of plasma was recorded, which points to a rapid onset of effect and a potential antioxidant benefit of the extract in the human body.

Metabolism of punicalagin

The results showed that after oral administration punicalagin was quickly metabolised to ellagic acid, which was then absorbed and conjugated. The conjugated form of ellagic acid was present in plasma at concentrations 5 to 8 times higher than the free form, regardless of the dose administered. UA appeared in the blood with a time lag, around 8 hours after administration, which supports the hypothesis of its gut origin mediated by the microbiota.

Conclusion

Given the growing interest in the role of the gut microbiome in the bioactivation of ellagitannins into UA, as well as their link to positive health effects, these results provide evidence of the functional activity of the polyphenols contained in the standardised pomegranate extract.

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