Nutrient Uptake Treatment with Postbiotics for Improved Functionality

The global scenario concerning undernutrition presents a critical challenge to public health, manifesting profound repercussions on morbidity and mortality worldwide. A notable manifestation of this issue is protein-energy undernutrition, wherein an individual's intake of essential macronutrients and proteins falls below the levels necessary for maintaining optimal health. 

This condition significantly contributes to the burden of various diseases on a global scale. In regions with abundant food resources, protein-energy undernutrition has become alarmingly prevalent among specific demographics, particularly affecting the elderly and individuals grappling with conditions that impair appetite, digestion, absorption, and nutrient metabolism. 

Chronic illnesses such as cancer or autoimmune disorders, infections, inflammatory conditions, metabolic disorders, and adverse drug reactions further exacerbate the prevalence of undernutrition in these populations. Indeed, Disease-Related Malnutrition (DMR) and micronutrients deficit are frequent conditions in the Inflammatory Bowel Disease (IBD) population reaching a prevalence of 23% in a recent multicentric study.

The mechanisms underlying malnutrition in IBD include reduced oral food intake due to loss of appetite, nausea, vomiting, abdominal pain, and diarrhea; malabsorption of nutrients due to leaky intestinal barrier, impaired epithelial transport, and loss of epithelial integrity; accelerated gastrointestinal transit; drugs-related micronutrients absorption and increased energy requirements due to systemic inflammation and inflammatory mediators synthesis.

The intricate interplay between undernutrition and health becomes even more pronounced with the profound alteration of intestinal structure and function. This alteration leads to impaired nutrient absorption and digestion, creating a ripple effect on associated medical conditions. The detrimental impact of inadequate nourishment extends beyond nutritional deficiencies, adversely affecting intestinal health.

The disruption of nutrient balance in the gut lumen compromises the maintenance of the intestinal barrier, leading to a cascade of effects. This includes the atrophy of intestinal villi, diminished protective mucus layer, dysbiosis, bacterial retention, and alterations in mucosal immune system functions. Collectively, these factors contribute to damage in the intestinal barrier, characterized by a reduction in tight junction proteins and increased gut permeability.

This compromised intestinal barrier, in turn, allows luminal content to leak into the submucosal space and circulation, thereby promoting both intestinal and systemic inflammation. This creates a vicious circle as inflammation further reduces appetite and increases protein consumption due to the synthesis of inflammatory mediators and to failed attempts of tissue repair. The multifaceted nature of the issue calls for holistic approaches, and the development of innovative technologies and models. 

The mentioned food supplement, which is based on a novel class of natural products, namely postbiotics, i.e. essential metabolites released during the metabolic activity of bacteria capable of conferring a beneficial effect to the host in either direct or indirect way, represents a promising step towards addressing the complex challenges posed by undernutrition on a global scale. Postbiotics comprise a multitude of metabolites (around 100) released during the fermentation process, ensuring multiple functional activities from intestinal barrier protection, anti-inflammatory activities, and microbiota reshaping.

The project employs an advanced methodology for the development of a novel postbiotic. Utilizing the fermentation process of Lactobacillus paracasei CNCM I-5220 through the application of our registered PBTech® fermentation method, to obtain a postbiotic formula that will be complemented with carefully curated blend of B-group vitamins and minerals. The production process comprises two phases: optimization of postbiotic production for yield and quality in a reproducible manner, and formulation of the best nutritional blend supporting protein-energy undernutrition. 

To offer meaningful insights into the feasibility of supporting patient’s unique nutritional needs, a preclinical experimental approach requires the utilization of distinct murine disease models that accurately replicate these specific requirements and evaluate the potential effects at the molecular level, through a combination of metabolomics, lipidomics, and microbiome sequencing, which could precisely identify the altered mechanisms underlying this multifactorial disease. Undernutrition models are typically induced by deficient diets because they are often considered similar to human conditions, as acute malnutrition is primarily associated with dietary deficiencies. For this purpose, we decided to perform an experimental study in mice feeding them with protein-deficient diets.

To unravel the biochemical mechanisms behind both low-protein and postbiotic-based food supplementation, the project will leverage on advanced metabolomics and lipidomics methodologies. Specifically, we will employ state-of-the-art mass spectrometry platforms dedicated to analyzing the polar metabolome and lipidome in plasma and feces. 

These platforms will utilize hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RP), coupled with high-resolution mass spectrometry (HRMS)14,15. With a small and efficient amount of starting material of 20 microliters of plasma and 10 milligrams of stool, these platforms enable the identification and relative quantification of over 800 analytes. 

This comprehensive analysis covers polar metabolites such as amino acids, organic acids, acylcarnitines, sugar phosphates, and nucleotides, as well as lipids spanning more than 40 subclasses. These robust methods will provide a detailed insight into the Winnie mice, allowing us to decipher how the new postbiotic-based formulation actively improves the health status of mice and modulates biochemical pathways. 

Furthermore, these data hold potential for translational applications of the novel postbiotic-based food supplement. In this context, the proposed project advocates for the implementation of an OMICS approach. This involves integrating 16S microbiota sequencing, along with metabolomic and lipidomic analyses. The objective is to gain a comprehensive understanding of how microbiota and consequently the associated metabolomic profile are altered in different undernutrition models and how, the intervention with a postbiotic-based food supplement can potentially restore both eubiosis and the proper permeability of the intestinal barrier; alleviating gut and systemic inflammation, reinstating the host's ability to effectively digest, metabolize, and absorb nutrients.

The development of a metagenomic tool utilizing Next-Generation Sequencing (NGS) technologies is key for understanding the impact of postbiotics on gut microbiota. Through 16S rRNA sequencing of fecal samples, Fem2-Ambiente aim to delve into microbial diversity, community structure, and differential abundance, providing valuable insights into the effects of experimental conditions. This metagenomicanalysis serves as a crucial component in unravelling correlations between bacterial composition and the observed outcomes, integrating seamlessly with other project data such as biological, metabolomic, and immunological parameters. 

The application of machine-learning approaches in analyzing microbiome data enhances our ability to predict sample groups, dietary conditions, and biological variations, thereby paving the way for innovative developments in postbiotic research. Furthermore, the microbiological analysis spearheaded by Fem2-Ambiente goes beyond project-specific objectives. By offering microbiota studies as a service, Fem2-Ambiente contributes to the broader community, fostering regional impacts.

This initiative not only advances scientific understanding but also establishes a platform for comprehensive microbiome profiling, thus ensuring that societal impacts are multifaceted, ranging from improved health outcomes to the promotion of scientific expertise and services within the community.

1. Innovative postbiotic formulation - capable of enhancing protein absorption will be developed and will achieve pre-clinical validation on models that are relevant to the raising undernutrition problems related to protein malabsorption. The postbiotic was produced through the FOS fermentation process and supplemented with functional components to improve quality of life in patients, reduce disease activity, and lower Calprotectin levels.
    
2. Murine reliable models will be established for dietary-induced and disease-associated protein-energy undernutrition. The model involved administering a low-protein diet to induce a moderate level of malnutrition, enabling the assessment of the supplement's therapeutic effects and its ability to mitigate damage caused by protein-energy undernutrition.
   
   Experimental groups included: Standard diet + vehicle (SD+V); Low Protein diet + vehicle (LPD+V); Standard diet + Postbiotic (SD+P); Low Protein diet + postbiotic (LPD+P). Results support the functional properties of the developed postbiotic-based dietary supplement in maintaining intestinal epithelial integrity in mice receiving a Low Protein Diet.
    
3. Develop a metagenomic tool using NGS (Next-Generation Sequencing) technologies to assess the effect of postbiotics. Perform statistical analyses to establish correlations between microbial diversity and experimental conditions, as well as variations in biomarkers detected by studies conducted by other project partners. 
   
   The postbiotic administration acted as a modulator selectively influencing certain taxa such as Muribaculaceae and Akkermansia.The increased abundance of Akkermansia and Bifidobacterium in standard diet and/or postbiotic groups suggests enhanced epithelial integrity and metabolic balance, whereas LP diet was associated with reduced butyrate producers and higher ecological instability. Co-occurrence analysis confirmed profound restructuring of ecological relationships, reflecting adaptive shifts toward new functional community configurations.
    
4. Results of the innovation will fill the knowledge gaps on treatment of undernutrition by evaluating the effect of a novel postbtiotic-based food supplement. Taken together, these results support the translational readiness of the postbiotic-based supplement formulation, which can now be positioned at a Technology Readiness Level (TRL) 5–6. 
   
   The formulation is safe, stable, and based on well-characterized components with established regulatory status (GRAS/QPS), making it suitable for further development toward pilot human studies. Its potential application extends beyond the treatment of undernutrition, encompassing broader clinical and nutritional contexts such as frailty, inflammatory bowel diseases, and metabolic disorders associated with gut barrier dysfunction.