It is well acknowledged that food is essential, but its vital role in the population’s health is often undervalued. However, this mindset has been slowly changing, as industry, researchers, and consumers are, nowadays, giving more attention to disease prevention and management through healthier diets, food safety, general well-being, and, more recently, the choice for more sustainable products and food production systems. The evident impact of food on human wellness has driven the urge to establish healthier dietary habits to fulfill food’s principal functions: supplying necessary nutrients, providing satisfaction, improving well-being, and regulating personal physiological states ^[1].

The search for food and food ingredients that provide additional benefits to consumers, called functional ingredients, that support or are supported by eco-friendly sustainable practices such as the reutilization of agro-industrial byproducts has been an area of increasing interest within the food industry and, recently, among consumers themselves ^[1]. Functional food additives are popular among consumers because of their improved organoleptic properties ^[2]. The development and commercialization of new functional ingredients to be incorporated in different food matrices now has a notorious impact on modern society’s dietary practices. In fact, ingredients such as probiotics and antioxidants are advertised as health promoters as a marketing strategy for food industries advocating the health benefits of their products (e.g., yogurts and fruit juices supplemented with probiotics and antioxidants, respectively).

The definition of functional food has been changing over the last few years; however, there is still no universally accepted single definition. Different international groups related to dietetics and nutrition (e.g., the International Food Information Council, the European Commission, and the American Dietetic Association) agreed that “functional food provides health benefits beyond basic nutrition” ^[1][3]. The international standards and guidelines for the evaluation of functional food are stipulated in the “Guidelines for use of nutrition and health claims (CAC/GL 23-1997)” of the Codex Alimentarius. When the established criteria are met, this codex allows the food sector to label its products with recognized health claims ^[4].

Functional foods are categorized into three classes based on their preparation: (1) conventional foods, (2) modified or fortified foods, and (3) food ingredients. Conventional foods are whole and unmodified foods (e.g., vegetables, meat, and cereal grains); fortified foods are regular foods supplemented with functional food components (e.g., calcium-fortified milk, anthocyanin-fortified bread, and vitamin-fortified honey); food ingredients are components from plants, microorganisms, and other inorganic raw materials that can be macronutrients, essential micronutrients, or non-nutrient components (e.g., inulin, Lactobacillus, iron) ^[1].

Overall, functional foods are meant to provide essential nutrients which can potentially bring additional health benefits to the host (e.g., stimulate the host’s immune system to prevent and control pathogenic infection) ^[3]. In the context of gut health promotion, prebiotics and probiotics are good examples of functional ingredients incorporated into food matrices. Prebiotics are substrates that are preferentially used by microorganisms within the host to provide health advantages to the host, whereas probiotics are live microbes that, when administrated in adequate quantities, deliver a health benefit to the host ^[5][6]. Numerous studies have helped us to understand the microorganisms’ functions and their beneficial impact on human health. Research with microorganisms, such as Lactobacillus, Bifidobacterium, Saccharomyces, and others, has made clear that their probiotic properties beneficially impact the gut’s health ^[7]. Other classes of food ingredients, such as polyphenols, polyunsaturated fatty acids (PUFAs), and phytochemicals have also been used to promote gut health ^[8].

2. Gut Microbiota—A Perspective on Fortified Food Properties

Nowadays, it is generally accepted that microbiota refers to the entire collection of microorganisms (e.g., bacteria, archaea, viruses, fungi) existing in a specific location, while microbiome refers to the collection of all genetic material within the microbiota. Gut microbiota includes all populations of beneficial (symbiotic) and/or harmful (pathogenic) microorganisms inhabiting the host’s gut, and the microbiome is the genome of all these microorganisms ^[9]. The microbial populations living in human gut microbiota are diverse (e.g., bacteria, archaea, and eukaryotes), abundant (from 10¹⁰ to 10¹² live microorganisms per gram in the colon), and in a close relationship with the host ^[9][10]. Bacillota (formerly Firmicutes) and Bacteroidota (formerly Bacteroidetes) are the main phyla, followed by Pseudomonadota (formerly Proteobacteria) and Actinomycetota (formerly Actinomycetes). These four phyla represent 93.5–98% of the bacteria in the gut microbiota, and common genera include Bifidobacterium, Lactobacillus, Bacteroides, Clostridium, and Escherichia ^{[11][12][13][14]}. The progress of human gut microbiota studies enabled the understanding that everyone’s microbiota is unique and develops from early childhood to adulthood. In adulthood, it is relatively stable and does not go through significant changes; however, it is still susceptible to change ^[15]. The gut microbiota is important for the host’s gut health and general well-being, as it has functions related to gut development, mucosal immunity and other immune system interactions, food digestion, nutrient absorption, body detoxification, and the production of important metabolites such as short-chain fatty acids (SCFA), arginine, glutamine, vitamin K, and folic acid ^[16][17]. The microbiota composition is influenced by genetic and environmental factors. Environmental factors are extrinsic to individuals and can be controlled or changed during the life of individuals. Examples of these include geographical localization, toxin/carcinogen exposure, bacterial infections, antibiotic treatment, lifestyle, surgery, and diet ^[15]. Several research papers argue that the nutritional value of foods is partially affected by the composition of the individual’s gut microbiota and that, in turn, food shapes the composition of the microbiota ^[15][18][19]. It is currently unknown what classifies a “healthy” microbiota, but it is acknowledged that about 30 to 40% of the adult human gut microbiota can be modified during its lifetime and the factor with the greatest impact, accounting for more than 50% of such variations, is diet ^[15][18]. Daily, humans consume foods that enhance the activity of indigenous bacteria of the gut microbiota such as fermented milk, processed cheeses, and yogurts that support the delivery of probiotics to the GIT ^[19]. Dysbiosis and eubiosis are concepts related to gut microbiota health that are still not fully understood and not clearly defined, although in recent years, researchers have agreed on the definitions of these concepts ^[20][21]. Dysbiosis is a term used to describe an unhealthy state of the gut microbiota, referring to any variation in the populations and functions of the resident commensal microorganisms in comparison with the populations of resident commensal microorganisms observed in healthy people ^[20][21][22]. Dysbiosis can be caused by stress, medical interventions, diet, and external factors ^[21]. Eubiosis is a term used to describe the opposite of dysbiosis, that is, a “balanced” microbiota found in healthy individuals ^[20]. These two concepts are quite vague and possess little scientific value since the composition of microorganisms inherent to the gut microbiota of healthy individuals is highly variable. Even if all species of microorganisms and their genes were cataloged, it would not illustrate how a healthy microbiota community is ^[20][22]. Although dysbiosis is also an imprecise term, different types of dysbiosis have been distinguished: (a) growth of pathobionts (commensal microorganisms that have the capacity to cause pathology), (b) loss of biodiversity, (c) loss of commensal microorganisms, (d) loss of beneficial microorganisms, and (e) shifts in microbiota metabolic capacity ^[20][22]. Currently, the food, nutrition, and pharmaceutical industries struggle to translate the results of experiments related to the human microbiota, mainly due to the absence of a clear scientific definition of what a “healthy” microbiota is ^[20]. Probiotics and prebiotics are good examples of concepts with clear definitions nowadays; however, these definitions emerged only after being discussed for years among the scientific community ^[5][6]. Thus, the absence of a clear definition demonstrates a lack of scientific rigor in this research area, which can hamper progress, making it difficult to discuss the concepts, disseminate information into general society, and apply that information to industry ^[20][21][22]. There is an urgent need and interest for reproducible, reliable, cost-beneficial, and fast laboratory techniques to assess and describe the state of the microbiota before and after food intake. Different types of foods differently impact the gut microbiota, and there are diets that may be beneficial or harmful to the microbiota, consequently affecting the host’s health. Examples of these unhealthy practices are the consumption of low-fiber, high-fat diets (Western diet) containing processed foods, while diets rich in fiber are usually related to a healthier state ^[21]. Advances in gut microbiota research are often dependent on the development of new techniques, technologies, and methodologies. At the moment, several techniques and technologies are used in the study of probiotics and gut microorganisms which enable research into microbial genes, transcripts, and proteins, providing information that be applied in food science studies ^[23]. There has been an increase in articles that highlight the importance of the intestinal microbiota and its fundamental role in the development of innovative strategies for the prevention and treatment of human health conditions such as obesity, gastrointestinal illnesses, inflammatory conditions, and psychiatric disorders, among others ^[21]. In the last ten years, medical research estimated an investment of USD 1.7 billion in the human microbiome research field, which augmented the “microbiome market” and the private investment of companies/start-ups in a large scope of food, pharmaceutical, and cosmetic products ^[9]. The modulation of the gut microbiota through a specific ingredient or a fortified food is then of the greatest importance for the evaluation of the true impact that each fortified formulation may have on consumers. Studies at the gut microbiota level allow the evaluation and prediction of several possible outcomes when adding functional ingredients to food matrices. Methodology related to the study of functional ingredients derived from agro-industrial byproducts and their impact on the gut microbiota is crucial, as most studies focus on the sensory and physicochemical properties of supplemented foods rather than the bioactivity potential of enhanced food matrices. The interaction of these ingredients with the gut microbiota can highlight the possible benefits to health from their supplementation to foods.

3. Methods Available to Evaluate Human Gut Microbiota Modulation

Nowadays, studies related to the human gut microbiome are one of the most dynamic research fields of science.  The use of different omics techniques (e.g., culturomics, metagenomics, transcriptomics, metatranscriptomics, proteomics, metabolomics) in the microbiology field have allowed to identify microorganisms and their metabolites, expanding our understanding of the impact that these microorganisms and their metabolites have on nutrition and health ^[9][23][24]. Examples of techniques used to study gut microbiome/microbiota are described in Table 13. The current challenges facing the food industry in the search for new functional ingredients to be added to food matrices can be overcome by combining individual omics techniques and obtaining basic insights regarding the effects of different compounds ^[23]. The common methodology, considered the “gold standard” for verifying bacterial viability in the food safety field, relies on culture-dependent methods ^[25][26]. However, culture-dependent methodology has its limitations, such as an inability to quantify uncultured bacteria, which represent around 60 to 80% of the bacterial populations present in gut microbiota ^[27][28]. The development of metagenomics techniques, such as quantitative polymerase chain reactions (qPCR), assists in overcoming these limitations, providing useful tools for the identification, description, and understanding of the role of each bacterial group in the microbiota ^[27][29]. But even these metagenomics techniques have their own limitations, as reviewed by Fraher et al. in 2012 and Shang et al. in 2018 ^[30][31]. Therefore, culturomics and metagenomics can be considered as techniques that complement each other and that can overcome each other’s limitations, depending on the proposed experimental design and the objective of the work. One of the omics that is extensively used in food science is metabolomics, which is a set of techniques for studying the metabolic pathways of biological systems by detecting and quantifying the production and/or changes of metabolites stimulated over time by biological systems (e.g., microbial communities) ^[23][32]. The food industry has a keen interest in analyzing the potential impacts of foods and food additives on metabolites produced by the gut microbiota, such as SCFA and especially butyrate, as these metabolites have a direct impact on the psychological state of the host ^[23]. Metabolomics analysis comprises two main approaches: directed analysis (determination and quantification of a group of pre-defined metabolites) and metabolic profiling (detection of all metabolites and/or their products) using a particular technical analysis accompanied by an absolute or relative quantity estimative and relying on chromatography techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) to separate compounds to be identified and quantified ^[24][32]. In most of the human microbiota (models or microbial mixed communities) impact studies, a combination of metagenomics and metabolomics techniques are used, with some studies using culture-dependent techniques to complement their experiments and collect valuable data to determine the impacts of specific compounds on microbial communities ^[24][27][33].