For decades, forensic science has relied on genetic markers for individual identification. However, as scientific understanding evolves, epigenetic markers—especially DNA methylation—are emerging as a significant focus. These markers not only help determine tissue types and estimate biological age, but also provide insights into a person’s lifestyle and environmental influences. This review delves deeper into the concept of epigenetics, its underlying mechanisms, and its current and potential applications in forensic investigations. It also examines the present methodologies, possible challenges, and the future prospects of this advancing field.
Introduction
Since the advent of DNA fingerprinting, forensic genetics has advanced considerably. While traditional techniques such as short tandem repeat (STR) analysis remain effective for identifying individuals, they are limited in providing insights into a person’s age, lifestyle, or environmental exposures. This is where epigenetics becomes relevant. Epigenetics involves modifications in gene expression that do not change the underlying DNA sequence. Among these, DNA methylation has emerged as the most promising for forensic applications, thanks to its stability and reproducibility. This paper explores the concept of epigenetic markers and highlights their expanding role in modern forensic science.
2. Epigenetics
Epigenetics
2.1 What is Epigenetics?
Epigenetics refers to chemical modifications that attach to DNA or its associated proteins, influencing how genes are activated or silenced. The most extensively studied of these modifications is DNA methylation, which involves the addition of a methyl group to the DNA, typically at a CpG site. Other forms of epigenetic modifications include histone changes and non-coding RNAs, but at present, DNA methylation is the most relevant and promising in the field of forensic science.
2.2 Variability in Epigenetic Markers
Epigenetics refers to chemical modifications that attach to DNA or its associated proteins, influencing how genes are activated or silenced. The most extensively studied of these modifications is DNA methylation, which involves the addition of a methyl group to the DNA, typically at a CpG site. Other forms of epigenetic modifications include histone changes and non-coding RNAs, but at present, DNA methylation is the most relevant and promising in the field of forensic science.
3. DNA Methylation in Forensics
3.1 Identifying Tissue Types
Different tissues have distinct methylation signatures. By examining these, experts can tell if a sample is blood, semen, saliva, menstrual blood, vaginal secretions, or skin cells. This information is vital in cases like sexual assaults or when dealing withmixed biological samples.
3.2 Estimating Age
Recent advances now allow forensic experts to estimate a person’s age by analyzing methylation levels in specific regions of the DNA. Models such as Horvath’s epigenetic clock can predict an individual’s age with an accuracy of just a few years, offering a valuable tool in cases where other identifying information is unavailable.
3.3 Indicating Lifestyle and Environmental Exposure
Emerging research indicates that methylation patterns can reveal aspects of an individual’s lifestyle, such as smoking, alcohol consumption, stress levels, and exposure to environmental toxins. Although this is still a developing area, these insights have the potential to offer valuable leads in forensic investigations.
4. Epigenetic Profiling
4.1 Preparing the Sample
The process starts by extracting DNA and treating it with bisulfite. This treatment helps differentiate between methylated and unmethylated cytosines, which is keytomethylation analysis.
4.2 Techniques Used
Pyrosequencing
Methylation-specific PCR (MSP)
Illumina BeadChip arrays
Next-generation sequencing (NGS)
Each method has its pros and cons in terms of accuracy, cost, and suitabilityfor degraded or limited samples.
5. Challenges and Concerns
5.1 DNA Quality and Quantity
For reliable results, relatively good-quality DNA is needed, which can be hardtocome by at crime scenes.
5.2 Variability Across Populations
Epigenetic patterns can differ significantly between individuals and population groups, which can sometimes affect accuracy.
5.3 Privacy and Ethical Issues
Predicting traits like age or habits based on DNA brings up ethical questions. Wheredo we draw the line between helpful evidence and invasion of privacy?
6. Scope of epigenetics
6.1 Need for Standardized Procedures
To make this procedure reliable in court, consistent procedures and validation across labs are crucial.
6.2 Blending with Traditional Methods
Epigenetic data can complement existing DNA profiling methods to build stronger forensic evidence.
6.3 Building Better Databases
More extensive epigenetic databases will help fine-tune prediction models andimprove their reliability.
7. Conclusion
Epigenetic markers are bringing a fresh wave of innovation to forensic DNAanalysis. DNA methylation, in particular, adds depth to what we can learn frombiological samples. Despite some hurdles like standardization and ethical concerns, the progress so far points toward a future where forensic epigenetics could become a standardpart of investigations.
The role of plants in human health and healing has been unmatched throughout the history of civilization. From the earliest forms of traditional medicine to the most cutting-edge discoveries in modern pharmaceuticals, plants have served as an invaluable source of healing compounds. Among these, phytochemicals — natural chemical compounds produced in plants — have emerged as particularly significant. These compounds possess remarkable anti-inflammatory, antioxidant, and antimicrobial properties. They are demonstrating exceptional potential in the prevention and treatment of a wide range of serious health conditions, including cancer, diabetes, cardiovascular diseases, and liver disorders. Moreover, ongoing research continues to unveil the diverse therapeutic effects of phytochemicals, further cementing their status as vital agents in promoting human health and well-being. As we continue to explore the intricate relationship between plants and health, it becomes increasingly clear that harnessing the power of phytochemicals holds tremendous promise for the future of medicine.
Phytochemicals—including flavonoids, terpenoids, saponins, alkaloids, and many others—exert a wide array of physiological and pharmacological effects on the human body. These naturally occurring compounds, present in plants, have garnered significant scientific interest due to their therapeutic potential. For example, codeine, a well-known analgesic, is derived from the opium poppy (Papaver somniferum) and is extensively utilized for its pain-relieving properties. Similarly, curcumin, the principal active component of turmeric (Curcuma longa), has gained global recognition for its potent anti-inflammatory and anticancer activities. Extensive research on curcumin has demonstrated its efficacy in modulating various cellular pathways, including the reduction of fat accumulation and the enhancement of cellular metabolic processes. These actions contribute to its potential role in the prevention and management of cardiovascular and hepatic diseases. Moreover, curcumin has shown promise in mitigating oxidative stress and inflammation, which are underlying factors in the pathogenesis of numerous chronic diseases. The study of phytochemicals thus continues to open new avenues for developing novel therapeutic agents and nutraceuticals aimed at improving human health and well-being.
The increasing body of research on phytochemicals in the field of medicine has significantly contributed to the development of innovative treatments and therapeutic approaches. Many contemporary pharmacological drugs are now being designed and developed by leveraging the bioactive compounds naturally present in plants, which often exhibit fewer side effects and enhanced efficacy compared to synthetic alternatives. For instance, ginseng—a well-known adaptogenic herb traditionally used to combat fatigue and enhance physical stamina—has demonstrated remarkable potential in both clinical and commercial pharmaceutical applications. Its active components, such as ginsenosides, have been shown to modulate energy metabolism and improve overall vitality, thereby assisting patients in managing fatigue-related conditions. These plant-based medicines offer a more holistic and sustainable approach to healthcare, promoting patient well-being in a safe and long-lasting manner. The integration of phytochemicals into modern drug development not only broadens the scope of treatment options but also reinforces the importance of natural resources in advancing human health.
Botany also plays a pivotal role in indigenous and traditional medicine systems across the world. In India, for instance, the foundations of Ayurvedic and Unani medicine are deeply rooted in the use of plant-based remedies. These traditional medical systems harness the therapeutic properties of various botanicals to address a wide spectrum of health conditions, often without causing significant side effects. For example, Ashwagandha (Withania somnifera), a revered medicinal herb in Ayurveda, is widely recognized for its adaptogenic properties, which help alleviate stress, combat fatigue, and enhance both mental and physical health. This synergy between botany and medicine not only provides effective therapeutic interventions but also plays a crucial role in the prevention of diseases by promoting overall well-being and resilience against stress-related disorders. The continued study and integration of traditional botanical knowledge into modern healthcare frameworks highlight the enduring importance of plants in enhancing human health and quality of life.
Recent research has revealed that certain phytochemicals play a crucial role in protecting human genetic material and preventing DNA damage. This groundbreaking discovery is opening new avenues for the prevention and treatment of cancer, as well as various genetic disorders. For instance, silymarin, a flavonolignan complex derived from milk thistle (Silybum marianum), has shown considerable promise in promoting liver health by protecting hepatocytes from toxins and facilitating tissue regeneration. Such findings underscore the value of integrating botanical and medical research to develop innovative therapeutic approaches. Furthermore, plant-derived compounds have demonstrated efficacy in the treatment and management of a wide array of chronic diseases, including arthritis, diabetes, cardiovascular disease, and more. This highlights the immense potential of natural products in expanding the modern pharmacopoeia, offering safer and often more holistic solutions to complex health challenges. The synergistic collaboration between botanical sciences and modern medicine thus continues to play a vital role in advancing human health and well-being.
With the use of these plant-based medicines, patients not only receive effective treatment but also experience significant improvements in their overall lifestyle and well-being. Consequently, the future of phytochemicals in the medical field is exceptionally promising, capturing the interest of both the general public and scientific communities alike. Modern medical research increasingly confirms that phytochemicals derived from natural plant sources tend to be safer and more beneficial for human health compared to many synthetic drugs and potentially harmful chemicals. This advantage is largely due to their biocompatibility, lower risk of adverse side effects, and their ability to work synergistically with the body’s natural processes. Therefore, further research and development in this field are imperative to establish phytochemicals as a robust and reliable therapeutic option. By deepening our understanding of the mechanisms of action, optimizing extraction and formulation methods, and conducting rigorous clinical trials, the integration of phytochemicals into mainstream healthcare can pave the way for more holistic, effective, and sustainable approaches to disease prevention and management in the future.
It will always be immensely beneficial to understand and utilize this rich treasure trove of plants to support and enhance our health. The integration of medicine and botany not only paves the way for innovative treatment approaches but also significantly advances the prevention of diseases and promotes an overall improvement in quality of life. By harnessing the therapeutic potential of phytochemicals and other plant-derived compounds, we can develop more holistic, safe, and effective healthcare solutions. This collaboration between natural and modern medicine empowers both patients and healthcare providers to embrace a more sustainable and patient-centered approach to wellness. As research continues to unveil the extraordinary potential of plant-based therapies, the synergy of medicine and botany will remain a cornerstone of human health and well-being.
The examination of biological fluids—namely blood, semen, and urine—is a cornerstone of forensic science, providing invaluable evidence during criminal investigations. These fluids can establish connections between a suspect and a crime scene, support or refute witness accounts, and aid in reconstructing the sequence of events surrounding an incident.
Blood analysis plays a pivotal role in forensic work by determining blood groups, developing DNA profiles for identification purposes, and detecting the presence of alcohol or drugs. Semen analysis is particularly significant in cases involving sexual assault, enabling forensic scientists to detect sperm cells and conduct DNA analysis to identify or eliminate potential suspects. Meanwhile, urine analysis is primarily used for toxicological investigations, revealing the presence of drugs, alcohol, or poisons that may have contributed to a crime or death.
This research focuses on the methods used to collect, preserve, and analyze these fluids, as well as the interpretation of their findings in a forensic context. Technological advancements, especially in DNA profiling, have substantially improved the accuracy and reliability of biological fluid examinations, solidifying their role as a fundamental component of modern forensic science.
Introduction
Contextual Information
Forensic science plays a crucial role in criminal investigations, as biological evidence often forms the foundation upon which cases are solved. Body fluids—including blood, semen, and urine—are among the most commonly encountered biological materials at crime scenes. These fluids are invaluable sources of information that, through scientific analysis, can reveal the identity of individuals and help reconstruct the events surrounding a crime.
Advancements in biotechnology have revolutionized the detection and analysis of biological fluids, allowing for the identification of even trace amounts with exceptional accuracy. Techniques such as DNA profiling, immunological assays, and sophisticated toxicological testing have significantly enhanced the reliability of forensic analyses. As a result, biological fluid examination has become a cornerstone of modern forensic science, offering critical evidence that can link suspects to crime scenes, support or challenge witness statements, and provide crucial insights into the circumstances of a crime.
Significance of the Topic
The examination of body fluids is essential in forensic investigations, as it can establish links between suspects and crime scenes, corroborate or refute alibis, and support the statements of victims and witnesses. Blood can provide crucial information such as DNA profiles, blood group analysis, and valuable insights through bloodstain pattern interpretation. Semen is particularly significant in cases of sexual assault, offering opportunities to identify perpetrators through the analysis of sperm cells and DNA profiling. Urine, on the other hand, is predominantly used for toxicological analysis, revealing the presence of drugs, alcohol, or poisons. The ability to analyze these fluids accurately can mean the difference between solving a case and a miscarriage of justice.
Scope of the Review
This review focuses on the forensic significance of blood, semen, and urine. It covers the methods used for the collection, preservation, and analysis of these biological fluids, as well as the interpretation of results and the influence of recent technological advances. Although the primary emphasis is on human biological fluids in the context of criminal investigations, comparative studies or findings from animal studies may be included when they are relevant to forensic practice.
Summary of the Main Findings in the Existing Literature
A substantial body of forensic literature underscores the critical role of biological fluids in crime scene analysis. Numerous studies consistently highlight that blood is the most commonly recovered biological fluid, with analytical methods ranging from presumptive tests (e.g., the Kastle-Meyer test) to confirmatory tests and DNA profiling. Research demonstrates that DNA extracted from dried bloodstains remains reliable for identification purposes even after several years, provided it is properly preserved.
Advancements in semen detection methods have also enhanced forensic investigations, particularly in sexual assault cases. Routine practices now include acid phosphatase testing, microscopic detection of sperm cells, and prostate-specific antigen (PSA) testing. Recent studies emphasize the high sensitivity and specificity of immunochromatographic tests while reaffirming the superiority of DNA analysis in establishing connections between suspects and victims.
Urine, though less commonly employed for direct identification, plays a crucial role in toxicological investigations. Several studies have utilized gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect scheduled drugs and poisons in urine samples, underscoring its value in determining the presence of substances that may influence behavior or contribute to death.
While there is general agreement regarding the effectiveness of DNA profiling in blood and semen analysis, some variability exists in the reported sensitivity and specificity of presumptive and confirmatory tests. For instance, a 2018 study by Sharma et al. reported that luminol testing can yield false positives in the presence of household cleaners, whereas a 2020 study by Wu et al. suggested that combining luminol with phenolphthalein improves accuracy.
In semen detection, some researchers advocate for the use of immunological tests alone due to their speed and specificity, while others stress the importance of microscopic sperm detection and DNA typing—especially in azoospermic cases where sperm cells may be absent.
Toxicological testing of urine samples also reveals differences in recommended detection windows and sample preparation methods. Some studies highlight the longer detection window of urine for drug use compared to blood, while others caution against potential contamination or misinterpretation caused by passive exposure or environmental sources.
Critical Evaluation of the Methods and Conclusions of the Reviewed Studies
Although the studies reviewed generally demonstrate robust methodologies, certain limitations are apparent. Many investigations are conducted under controlled laboratory conditions that may not accurately replicate real-world crime scene environments, where samples are subject to degradation from factors such as temperature fluctuations, humidity, and contamination. Furthermore, some earlier studies relied heavily on presumptive testing without sufficient confirmatory follow-up, which can compromise the reliability and forensic validity of their findings.
In semen detection, some studies did not account for cases of male infertility or vasectomy, where sperm cells may be absent, leading to potential false negatives if only sperm detection methods are used. Similarly, in urine testing, although highly sensitive techniques such as gas chromatography-mass spectrometry (GC-MS) are preferred, they are costly and require specialized equipment, potentially limiting their application in resource-constrained forensic laboratories.
Identification of Gaps or Inconsistencies in the Literature
There is a noticeable gap in the literature regarding the durability and detectability of biological fluids under various environmental conditions over extended periods. Few studies have systematically explored how factors such as temperature, humidity, and exposure to light or contaminants impact the degradation of biological markers critical for fluid identification. Additionally, there is an evident lack of standardization in testing protocols across regions and laboratories, which may affect the reproducibility and legal admissibility of results in court proceedings.
Moreover, there is limited research on the forensic significance of mixed biological specimens (e.g., mixtures of semen and urine), as well as on the chemical interference that might affect the detection and analysis of these fluids. Finally, an emerging and underexplored area is the integration of artificial intelligence (AI) and machine learning in the analysis of complex biological evidence datasets. This presents a promising avenue for future research, potentially enhancing the accuracy and efficiency of forensic analyses.
Result
Summary of the Findings from the Reviewed Literature
The literature reviewed indicates that each biological fluid—blood, semen, and urine—has unique forensic utility depending on the nature of the investigation. Blood is the most commonly recovered biological fluid, with well-established detection and profiling techniques that support its use in linking suspects to crime scenes and reconstructing events. Semen plays a critical role in sexual assault cases, offering strong evidentiary value due to the presence of sperm cells and high-quality DNA. Although urine is not typically used for identification purposes, it is highly effective for toxicological analyses and the detection of drugs and poisons.
Across multiple studies, DNA profiling has been identified as the most effective method for analyzing semen and blood, while techniques such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) are better suited for urine toxicology. However, variations in the accuracy of presumptive tests have been observed, often influenced by environmental conditions and the presence of contaminants.
forensic technique
Dicussion
Interpretation of the Findings
The evidence from the reviewed literature confirms that biological fluids—particularly blood, semen, and urine—are fundamental to forensic analysis. Blood remains the most valuable fluid, providing not only DNA for individual identification but also insights into crime scene dynamics through bloodstain pattern analysis. Semen is primarily linked to sexual offenses, offering abundant genetic material when sperm cells are present. While urine is less suitable for DNA profiling, it plays a crucial role in toxicological screening by detecting drugs and toxins.
These findings highlight that the forensic relevance of each fluid largely depends on the context of the investigation. Furthermore, the studies validate the effectiveness of advanced analytical techniques such as DNA profiling and gas chromatography, while also emphasizing that test performance can vary based on sample condition and environmental factors.
Implications of the Findings
The implications for forensic science are significant. Understanding the strengths and limitations of each bodily fluid allows forensic practitioners to choose appropriate analytical methods and interpret evidence with greater accuracy. In a legal context, reliable analysis enhances the evidentiary value of biological fluids and can profoundly influence case outcomes. Moreover, integrating biological fluid analysis with other forensic disciplines—such as toxicology, serology, and trace evidence examination—strengthens the overall investigative process.
Comparison to Other Studies
Compared to earlier forensic literature, the present review both reaffirms well-established findings and highlights advances in detection technologies. For example, older studies commonly relied on presumptive tests with lower specificity, whereas recent research emphasizes combining DNA analysis with immunological assays to achieve greater accuracy and reliability. This shift reflects the broader progression in forensic science toward more robust, confirmatory methods and a decreased reliance on solely visual or presumptive indicators.
Limitations of the Review
This review has several limitations. Firstly, it relies on secondary data from published studies that vary in methodology and quality. Many of these studies were conducted under controlled laboratory conditions, which may not accurately reflect real crime scene environments where factors such as environmental degradation can compromise sample integrity. Secondly, the review focuses exclusively on three primary fluids—blood, semen, and urine—while other biologically relevant substances like saliva, sweat, and vomit, which also hold forensic significance, were excluded.
Future Directions of Research
Future research should focus on improving the detection of biological fluids in degraded or mixed samples, particularly those collected from outdoor crime scenes or after significant time lapses. Investigating the effects of environmental factors such as temperature, humidity, and microbial activity on fluid preservation would be highly beneficial. There is also growing emphasis on standardizing testing protocols across forensic laboratories to ensure consistency and enhance legal admissibility. Furthermore, the integration of artificial intelligence and machine learning in analyzing complex forensic evidence—such as fluid identification and DNA mixture interpretation—presents a promising avenue for research and development.
Conclusion:-
Summary of the Main Points
This review has highlighted the forensic significance of three primary biological fluids—blood, semen, and urine—in criminal investigations. Blood plays a vital role in DNA profiling and crime scene reconstruction, while semen is especially important in sexual offense cases through sperm detection and genetic analysis. Although urine is less commonly used for identification, it remains essential for toxicological screening of drugs and poisons. The reviewed literature underscores the importance of proper collection, preservation, and analytical methods to maintain the integrity and reliability of these samples.
Restatement of the Importance of the Topic
Accurate identification and analysis of biological fluids remain the cornerstone of modern forensic science. These fluids provide crucial evidence that can establish links between suspects, victims, and crime scenes. With ongoing advancements in forensic technology, the role of biological fluid analysis will continue to grow in importance, playing an increasingly vital part in the pursuit of justice.
Final Thoughts and Recommendations
Moving forward, forensic practitioners are encouraged to adopt more holistic approaches that integrate traditional serological techniques with advanced DNA and toxicological analyses. Greater standardization of testing protocols, along with expanded research on environmental effects and sample degradation, is essential. Additionally, the incorporation of emerging technologies—such as AI-driven data analysis—will further enhance the accuracy and efficiency of biological fluid analysis in forensic science.
Humans are exceptional reservoirs of diverse microbial species, forming complex microbiota that play a crucial role in health by modulating metabolic processes and protecting against various diseases. The composition and function of the microbiota can be positively influenced by the consumption of probiotics and prebiotics—beneficial bacteria and non-digestible food components, respectively—that promote the growth of these beneficial microbes. Many fermented foods serve as sources of probiotic strains, while plant-based oligosaccharides are well-known prebiotics. Together, probiotics and prebiotics are important in treating immune system disorders, cancer, liver diseases, gastrointestinal issues, type 2 diabetes, and obesity, thanks to their immunomodulatory properties, support of gut barrier integrity, production of antimicrobial compounds, and regulation of immune responses. This review aims to highlight the potential impact of prebiotics and probiotics on gut microbiota, emphasizing their role in enhancing health benefits.
Introduction
The human microbiota contains approximately 101410^{14}1014 bacterial cells, which is comparable to the total number of eukaryotic cells in the human body (Wang et al., 2016). Although the gut of a neonate is initially sterile, it is rapidly colonized by maternal bacteria, resulting in a diverse gut microbiome. During the first few months of life, the infant’s gut microbiota adapts to its environment based on nutritional availability, anaerobic conditions, and microbial interactions (Bäckhed et al., 2015).
The diversity of the adult gut microbiota consists of approximately 1,000–1,150 different bacterial species, primarily including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia (David et al., 2014; Salvucci, 2019; Almeida et al., 2019). Lifestyle, environment, and age significantly influence the stability of the host microbiome (Di Paola et al., 2011).
Although the gut microbiota remains generally resilient and stable throughout a person’s life, it can be temporarily affected by factors such as unhealthy diets, antibiotic use, and exposure to new environments—though these usually have a limited impact on its overall composition (Rajilić-Stojanović et al., 2013).
As a result, prebiotics are selectively utilized by the host’s microbiota to confer health benefits (Swanson et al., 2020). Their potential effects include modulation of the gut microbiota and the production of beneficial metabolites, such as tryptophan and short-chain fatty acids (SCFAs). Commercially available prebiotics include inulin, isomalto-oligosaccharides (IMO), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose, and resistant starches (Yan et al., 2018).
Probiotics are live microorganisms that provide health benefits to the host when administered in adequate amounts. Common probiotic species include members of the Bifidobacterium and Lactobacillus genera, while less common probiotics include Faecalibacterium prausnitzii, Akkermansia muciniphila, Streptococcus thermophilus, Saccharomyces boulardii, and Lactococcus lactis (Hill et al., 2014; Markowiak & Śliżewska, 2017; Ballan et al., 2020). These probiotics influence the gut luminal environment, mucosal barrier function, and mucosal immune system.
Composition and diversity of gut microbiota
The human microbiota contains approximately 101410^{14}1014 microorganisms. The mother’s oral microbiota closely resembles that of the placenta, with Firmicutes, Proteobacteria, Bacteroides, Fusobacteria, and Tenericutes contributing to its structure during the prenatal period. According to one theory, microbes may be transferred from the oral cavity to the fetus via the circulatory system (Aagaard et al., 2014).
After birth, the newborn encounters various microbes, with colonization influenced by the mode of delivery (Dominguez-Bello et al., 2010). During vaginal delivery, the baby’s skin and mucous membranes come into contact with the mother’s microbiota, leading to colonization predominantly by Lactobacillus species. In contrast, Propionibacteria and Staphylococcus—common skin microbes—colonize the baby’s mouth, gut, and skin following cesarean section (Jakobsson et al., 2014; Bäckhed et al., 2015).
During the initial months of a newborn’s life, the gut microbiota adapts to its environment, influenced by factors such as nutrient availability, anaerobic conditions, and microbial interactions (Bäckhed et al., 2015). In the first two years, cesarean-born infants tend to have fewer maternally transmitted microbes (e.g., Bifidobacteria and Bacteroides), lower diversity, and a reduced Type 1 T helper (Th1) immune response compared to vaginally born infants; however, these differences gradually diminish over time (Jakobsson et al., 2014; Bäckhed et al., 2015).
Breastfeeding is another key factor in early microbial colonization. Human breast milk contains more than 10710^{7}107 bacterial cells per 800 mL, predominantly from the genera Lactobacillus, Streptococcus, and Staphylococcus (Soto et al., 2014). Additionally, breast milk is rich in oligosaccharides, which selectively promote the growth of Lactobacillus and Bifidobacterium species. These microbes contribute to fermentation in the gastrointestinal tract (GIT), the production of short-chain fatty acids (SCFAs), and the reduction of colonic pH. This acidic environment limits the growth of harmful pathogens that cannot survive under such conditions.
Furthermore, breast milk provides immunoglobulin A, lactoferrin, and defensins, which offer additional health benefits to infants (Lönnerdal, 2016). Early breastfeeding has also been linked to the prevention of diseases such as obesity, dermatitis, and infections (Greer et al., 2008).
Following the introduction of solid foods, the infant’s gut microbiota begins to transition toward an adult-like composition (Turnbaugh et al., 2009). By the third year of life, environmental factors strongly influence microbiota colonization. This increased diversity enhances the synthesis of vitamins and amino acids and improves carbohydrate metabolism (Yatsunenko et al., 2012).
In adulthood, the gut microbiota is typically composed of Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Proteobacteria. This diverse microbiota plays a critical role in human physiology, influencing intestinal barrier integrity, neurotransmitter production, immune system development, and energy metabolism. Lifestyle, physical environment, and age all affect the stability of the host microbiome (Di Paola et al., 2011).
Methodology
This review adheres to PRISMA guidelines and includes literature sourced from Google Scholar, PubMed, Scopus, and Web of Science. Over 50 peer-reviewed studies from the past decade examining the health benefits of probiotics and prebiotics were selected. Quality assessments were conducted using the Cochrane Risk of Bias tool for randomized controlled trials (RCTs) and the Newcastle-Ottawa Scale for observational studies.
The findings were synthesized narratively, focusing on three key areas: the role of gut microbiota in combating diseases, the role of prebiotics in modulating gut microbiota, and the role of probiotics in supporting gut microbiota.
Factors influencing the gut microbiota
To explore the connection between genetics and gut microbiota, researchers profiled the gut microbiota of eight distinct mouse breeds using DNA fingerprinting techniques. A previous study (Kemis et al., 2019) found that the host’s genetic makeup significantly influences microbiota diversity. The host genotype also plays a role in selecting the intestinal microbiota.
At birth, the newborn’s sterile gut is already colonized by numerous microbes from the mother and the surrounding environment. Although the formation of the gut microbiota is influenced by the offspring’s genes, mothers and their children share approximately half of their genetic material as well as similarities in their gut microbiota composition (Coelho et al., 2021).
The adult gut microbiota is highly responsive to dietary changes. In studies where mice were switched to a Western-style diet, the microbiota underwent significant alterations, notably with a marked increase in the abundance of Firmicutes, particularly the class Erysipelotrichi (Salazar et al., 2017). Changes in diet over just 24 hours triggered observable shifts in microbial composition.
Dietary carbohydrates that are indigestible in the upper intestine reach the colon where they are fermented by gut microbes, leading to substantial changes in microbiota composition and beneficial effects on host health (Leeming et al., 2019). The prebiotic hypothesis, first proposed in 1995, highlights that prebiotics can increase the number of Bifidobacteria (phylum Actinobacteria) (Rezende et al., 2021). This microbial shift happens quickly but also reverts rapidly once prebiotic intake stops.
The adult gut microbiota is highly responsive to dietary changes. In studies where mice were switched to a Western-style diet, the microbiota underwent significant alterations, notably with a marked increase in the abundance of Firmicutes, particularly the class Erysipelotrichi (Salazar et al., 2017). Changes in diet over just 24 hours triggered observable shifts in microbial composition.
Dietary carbohydrates that are indigestible in the upper intestine reach the colon where they are fermented by gut microbes, leading to substantial changes in microbiota composition and beneficial effects on host health (Leeming et al., 2019). The prebiotic hypothesis, first proposed in 1995, highlights that prebiotics can increase the number of Bifidobacteria (phylum Actinobacteria) (Rezende et al., 2021). This microbial shift happens quickly but also reverts rapidly once prebiotic intake stops.
Breast milk naturally contains oligosaccharides that act as prebiotics, supporting the growth of Bifidobacterium populations in infants. These findings emphasize the crucial role diet plays in shaping the gut microbiota throughout life (Leeming et al., 2019).
The body’s immune system plays a crucial role in shaping the gut microbiota. Studies have shown that animals with abnormal Toll-like receptor (TLR) signaling exhibit elevated antibody levels, which help regulate commensal bacteria. This interaction between the host and gut microbiota is maintained through increased serum antibody levels.
Mutant mice lacking functional TLRs display altered intestinal microbial compositions, demonstrating that the host’s phenotype is strongly influenced by the characteristics of its gut microbiota. Additionally, factors such as gut peristalsis and the dense mucus layer produced by goblet cells affect the microbial population (Schluter et al., 2020).
The thick mucus layer formed by goblet cells acts as a barrier, limiting microbial penetration into the colonic epithelium (see Figure 1).
Role of microbiota in combating diseases
The gut microbiota plays a key role in metabolic processes, including carbohydrate and lipid metabolism, which are critical factors in the development of diabetes. Certain probiotic strains can improve insulin sensitivity and help regulate blood glucose levels. For example, Lactobacillus and Bifidobacterium strains have been shown to reduce inflammation and enhance glucose metabolism, thereby alleviating the effects of type 2 diabetes (Turnbaugh et al., 2009).
These probiotics exert their beneficial effects by strengthening gut barrier function, lowering endotoxemia, and influencing the secretion of hormones such as incretins, which are involved in insulin release. Moreover, they can affect the gut-brain axis, potentially reducing stress-induced hyperglycemia and further supporting better glycemic control (Schluter et al., 2020).
Microbiota
Gut bacteria metabolize dietary compounds like choline and carnitine into metabolites that influence cardiovascular health. Probiotic strains such as Lactobacillus reuteri help lower cholesterol by breaking down bile acids in the gut, preventing their reabsorption and thereby reducing blood cholesterol levels.
Disruptions in the gut microbiome have been linked to cardiovascular diseases. Certain probiotics offer potential therapeutic benefits by modulating inflammation and reducing hypertension (Leeming et al., 2019). These probiotics also produce short-chain fatty acids (SCFAs), which have been shown to lower blood pressure and enhance endothelial function. Furthermore, they can reduce levels of trimethylamine N-oxide (TMAO), a metabolite associated with higher cardiovascular risk, thus providing a comprehensive approach to supporting heart health (Champagne et al., 2018).
The gut microbiome plays a crucial role in adipose tissue metabolism and the development of obesity. Research shows that the composition of gut microorganisms varies between underweight and overweight individuals. Specific probiotic strains, such as Lactobacillus gasseri and Bifidobacterium breve, influence energy balance and fat storage by regulating nutrient absorption and hormone secretion related to appetite and fat accumulation.
Moreover, short-chain fatty acids (SCFAs) produced by gut bacteria promote adipocyte differentiation, enhance lipid metabolism, and improve insulin sensitivity, all of which contribute to better metabolic health (Aagaard et al., 2014). These SCFAs act as signaling molecules that regulate gene expression involved in lipid metabolism and energy homeostasis. Probiotics also stimulate the release of satiety hormones like peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), which help decrease food intake and support weight loss (Schluter et al., 2020).
Emerging studies indicate that the gut microbiome significantly influences cognitive function and mental health via the gut-brain axis. A healthy gut microbial community is linked to a lower risk of mood disorders such as depression and anxiety. Probiotic strains like Lactobacillus helveticus and Bifidobacterium longum have demonstrated the ability to reduce stress and anxiety symptoms by modulating neurotransmitter production and lowering systemic inflammation (Turnbaugh et al., 2009).
These probiotics impact levels of serotonin and gamma-aminobutyric acid (GABA), both essential neurotransmitters for mood regulation. Additionally, they suppress pro-inflammatory cytokines that can adversely affect brain function. By enhancing gut barrier integrity, these probiotics help prevent inflammatory molecules from reaching the brain, thereby promoting mental well-being (Rosolen et al., 2019).
A diverse gut microbiota is essential for a strong immune system, aiding in the management of asthma and the reduction of allergies. Exposure to a broad range of microbes enhances immune resilience and lowers the risk of autoimmune diseases. Probiotic strains such as Lactobacillus rhamnosus can modulate immune responses and alleviate allergic symptoms by strengthening gut barrier function and decreasing pro-inflammatory cytokines (Aagaard et al., 2014).
These probiotics stimulate the production of regulatory T cells (Tregs), which help sustain immune tolerance and prevent excessive reactions to harmless antigens. They also boost the secretion of secretory IgA, a critical antibody in mucosal immunity, offering extra protection against allergens and pathogens (Schluter et al., 2020).
An imbalance in the gut microbiota is linked to several gastrointestinal disorders, including irritable bowel disease (IBD) and colitis. Managing the gut microbiota through dietary interventions and probiotics can help reduce inflammation and promote gut health. Probiotics such as Saccharomyces boulardii and Lactobacillus plantarum have demonstrated effectiveness in alleviating IBD symptoms and preventing relapses by enhancing mucosal barrier function and modulating immune responses (Leeming et al., 2019).
These probiotics help restore gut flora balance, decrease pro-inflammatory cytokine production, and increase anti-inflammatory cytokine levels. They also support regeneration of the gut epithelium and strengthen gut barrier integrity, thereby reducing intestinal permeability and subsequent inflammation (Champagne et al., 2018).
The gut microbiome plays a crucial role in modulating certain types of cancer, including colon cancer. Some gut bacteria elicit anti-inflammatory responses that may protect against tumor formation. Probiotics such as Lactobacillus casei have been shown to reduce the risk of colon cancer by inhibiting the growth of pathogenic bacteria and promoting the production of anti-carcinogenic compounds (Turnbaugh et al., 2009). These probiotics increase the production of butyrate, a short-chain fatty acid with well-known anti-tumorigenic properties. Butyrate induces apoptosis in cancer cells and inhibits their proliferation. Additionally, probiotics can modulate the immune system to enhance its ability to recognize and destroy cancer cells, providing a dual mechanism against tumor development (Rosolen et al., 2019).
The gut microbiota also influences liver health through several mechanisms. Dysbiosis, or imbalance of gut microbes, can increase intestinal permeability (“leaky gut”), allowing toxins to enter the bloodstream, trigger systemic inflammation, and contribute to liver disorders. Probiotics such as Lactobacillus rhamnosus help maintain gut barrier integrity and reduce liver inflammation. Moreover, gut bacteria participate in bile acid metabolism, essential for digesting dietary fats and regulating lipid and glucose metabolism in the liver (Aagaard et al., 2014). Probiotics can also decrease the production of lipopolysaccharides (LPS), endotoxins that promote liver inflammation and damage. By modulating gut microbiota composition and function, probiotics support liver health and help prevent progression of liver diseases such as non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (Rosolen et al., 2019) (see Figure 2).
Role of prebiotics in gut microbiota
Prebiotics are defined as substances selectively utilized by host microorganisms to confer health benefits (Swanson et al., 2020). These benefits include modulation of the gut microbiota and the production of metabolites such as short-chain fatty acids (SCFAs) and tryptophan derivatives. However, these effects should be confirmed in target hosts, including animals and humans (Roager & Licht, 2018; Sanders et al., 2019; Swanson et al., 2020). Common commercially available prebiotics include inulin, lactulose, fructo-oligosaccharides (FOS), isomalto-oligosaccharides (IMO), galactooligosaccharides (GOS), and resistant starch (Yan et al., 2018). Numerous studies have explored the health benefits of dietary fiber consumption, both with and without recognized prebiotic effects. The primary mechanism of prebiotics involves selective fermentation by beneficial gut microorganisms, such as Lactobacillus and Bifidobacterium, which produce acetate and lactate, respectively. These metabolites then stimulate other beneficial microbes to produce butyrate, a key SCFA. Importantly, SCFAs have been shown to enhance mineral absorption, contributing to improved host health (Roager & Licht, 2018; Sanders et al., 2019; Swanson et al., 2020).
Prebiotics can assist in regulating the overall bacterial diversity of the gut by promoting the growth of useful bacteria, while inhibiting the proliferation of potentially dangerous species. Prebiotics can modulate immune responses and reduce inflammation by influencing lymphoid tissue associated with the gut. Prebiotic consumption has been linked to a variety of health benefits, including improved digestive health, enhanced nutrient absorption, and a lower risk of certain chronic diseases such as obesity, diabetes, and cardiovascular disorders.
Health Benefits
According to Oliveira et al., co-cultures of probiotics with certain strains combined with inulin—the most extensively studied prebiotic—improve the acidification rate of dairy products. Santos et al. demonstrated that Lactobacillus acidophilus La-5, when microencapsulated with inulin, showed greater resistance to simulated gastrointestinal tract (GIT) stress in vitro compared to free cells, resulting in an enhanced survival rate (David et al., 2014). Additionally, Rosolen et al. reported that using a combination of whey and inulin as a protective coating for Lactococcus lactis R7 improved heat resistance and tolerance to in vitro GIT stress (see Table 1).
Role of probiotics in gut microbiota
Good health is strongly linked to the ingestion of probiotics. The microbes approved for consumption are generally considered safe, with selective strains targeting specific populations such as newborns, adults, and the elderly. Additionally, the recommended dietary allowance (RDA) of these microbes should be taken into account to achieve optimal health benefits (Hill et al., 2014; Ballan et al., 2020; Coniglio et al., 2023). Common probiotic species include those from the genera Lactobacillus and Bifidobacterium, while other microbes such as Faecalibacterium prausnitzii, Akkermansia muciniphila, Streptococcus thermophilus, Saccharomyces boulardii, and Lactococcus lactis are also categorized as probiotics (Hill et al., 2014; Markowiak & Śliżewska, 2017; Ballan et al., 2020).
Different probiotic strains exhibit varying survival and multiplication rates in the stomach depending on factors such as the food medium (e.g., milk or soymilk), oxygen levels (e.g., stirred yogurt), storage temperature, pH, and the presence of food ingredients or prebiotics (Homayoni Rad et al., 2016; Champagne et al., 2018; Ballan et al., 2020). By reducing certain unfavorable food components, such as raffinose and stachyose found in soymilk, probiotics can exert beneficial health effects (Albuquerque et al., 2017; Battistini et al., 2018; Champagne et al., 2018). While starter strains like Streptococcus thermophilus are added alongside probiotic cultures to shorten fermentation times, their presence can inhibit the production of flavors generated by acetic acid when co-cultured with Bifidobacterium strains (Tripathi & Giri, 2014; Oliveira et al., 2009; Champagne et al., 2018).
Prebiotics are often consumed together with probiotics to form synbiotics, which can reduce fermentation time and enhance the survival rate of probiotics throughout the gut (Oliveira et al., 2009; Markowiak & Śliżewska, 2017). Dietary changes modulate the behavior of probiotic strains differently. For example, the addition of fruit pulp to soymilk fermented with probiotics significantly influences the properties of the final product (Peters et al., 2019). Furthermore, advancing food technologies such as microencapsulation have greatly improved fermentation methods and increased tolerance to gastrointestinal tract (GIT) stresses (Oliveira et al., 2009; Champagne et al., 2018; Tripathi & Giri, 2014). Recent studies indicate that microbial metabolites contribute to health benefits and influence probiotic function and supplementation strategies (Champagne et al., 2018; Kalita et al., 2023; Mehmood et al., 2023) (Table 2).
Mechanism of action
Probiotic microorganisms influence the host in several ways, enhancing the intestinal lumen, mucosal barrier, and immune stability (Fong et al., 2020). These effects are mediated through various cell types involved in both innate and adaptive immunity, including epithelial cells, monocytes, dendritic cells, B cells, T cells (such as regulatory T cells), and natural killer (NK) cells. The primary mechanisms include selective utilization of prebiotics by commensal microbiota, production of metabolites like short-chain fatty acids (SCFAs) and organic acids, reduction of lumen pH, increased mineral absorption, and inhibition of pathogenic growth (Peters et al., 2019) (Figure 3).
Probiotics enhance phagocytosis, regulate immunoglobulin production, improve immune responses, and maintain microbiome homeostasis through competition for nutrients and adhesion sites, bacteriocin release, reduction of pro-inflammatory activities, and enhancement of barrier functions (Bermudez-Brito et al., 2012). Key regulatory pathways and cytokines involved include G protein-coupled receptors (GPR41 and GPR43), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), lipopolysaccharides (LPS), nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), exopolysaccharides (EPS), interferon-gamma (IFN-γ), and interleukin-12 (IL-12). These mechanisms play important roles in reducing metabolic endotoxemia and inflammation (Peters et al., 2019).
Moreover, probiotics modulate mucosal cell interactions and maintain cellular stability by improving intestinal barrier function. They achieve this by regulating the phosphorylation of cytoskeletal and junctional proteins, which supports barrier integrity through processes such as mucus production.chloride and water secretion, and tight junction protein interactions (Yadav & Jha, 2019).
Enhanced mucosal barrier function is crucial in managing disorders such as inflammatory bowel disease (IBD), celiac disease, gut infections, and type 1 diabetes (Ghosh et al., 2021). At the molecular level, epithelial cells respond differently to commensal or probiotic bacteria compared to pathogens. For instance, probiotic bacteria do not induce interleukin-8 (IL-8) secretion from epithelial cells, whereas pathogens like Shigella dysenteriae, enteropathogenic Escherichia coli, Listeria monocytogenes, and Salmonella dublin do (Bermudez-Brito et al., 2012). In fact, co-culture with….
Probiotic bacteria can reduce IL-8 release caused by these pathogens, thereby mitigating inflammation and promoting intestinal homeostasis. However, not all probiotics exhibit this anti-inflammatory trait; for example, Escherichia coli Nissle 1917 has been shown to increase IL-8 secretion in a dose-dependent manner, highlighting the variability in the immunomodulatory effects of different probiotic strains (Wen et al., 2020).
Future perspectives
Advancements in gut microbiome profiling tech-niques will enable personalized approaches to gut health interventions. By identifying an individual’s gut microbiota composition and its response to
probiotics_machanis
By leveraging prebiotics and probiotics, healthcare professionals can design targeted treatment strategies to maximize health benefits. Researchers continue to explore novel prebiotic and probiotic strains to optimize their effects on gut health. Advances in microbial engineering and genetic editing technologies have facilitated the development of more precise and potent prebiotics and probiotics, thereby maximizing their therapeutic potential (Wen et al., 2020).
The gut-brain axis—a bidirectional communication network linking the gut and the brain—illustrates how gut microorganisms influence mental health and cognition. This connection opens avenues for developing prebiotic and probiotic interventions aimed at supporting mental health and reducing symptoms of depression. Moreover, the therapeutic applications of prebiotics and probiotics extend well beyond gut health. Emerging research has examined their roles in managing metabolic disorders, cardiovascular diseases, and autoimmune conditions. Identifying specific microbial strains and bioactive compounds capable of modulating disease-related pathways offers promising new directions for targeted therapies (Peters et al., 2019).
Microbiome-based therapeutics, including fecal microbiota transplantation (FMT) and defined microbial cocktails, show considerable promise for treating gastrointestinal disorders and systemic diseases. As our understanding of the distinct functions of various microbial communities deepens, these therapies are expected to become increasingly refined and widely accepted in mainstream medicine. However, the rapid growth of prebiotic and probiotic products in the market has outpaced regulatory oversight, leading to concerns about inconsistent quality and efficacy. Enhanced regulatory frameworks will be essential to ensure the safety, reliability, and therapeutic value of these products.
Notably, early-life exposure to prebiotics and probiotics may have long-lasting effects on gut health and overall well-being. Understanding how these early interventions shape the developing gut microbiome—and consequently influence lifelong health—is a critical area of ongoing research (Rosolen et al., 2019).
As gut microbiota science advances, it is likely that nutritional guidelines will increasingly incorporate prebiotic and probiotic recommendations to promote gut health. Integrating these into standard dietary advice could help prevent gut-related disorders and improve general health outcomes. In conclusion, the future of prebiotics and probiotics in gut microbiota research is promising and holds vast potential to enhance human health. Continued exploration of the gut microbiome’s complexities and its broad impact on well-being will drive the development of personalized interventions and innovative therapeutics, revolutionizing approaches to gut health and disease management (Wen et al., 2020).
Limitations
The potential impact of probiotics and prebiotics in enhancing health benefits is promising, yet several limitations must be acknowledged. Much of the current evidence relies on studies with small sample sizes, short durations, or investigations focused on specific populations, thereby limiting the generalizability of findings. Additionally, the variability in probiotic strains, prebiotic compounds, dosages, and methodological inconsistencies across studies complicates the interpretation and comparison of results. Individual differences in microbiome composition and the complex interactions between probiotics, prebiotics, and host physiology are often oversimplified, further challenging the establishment of universal guidelines. Potential biases, such as industry sponsorship and publication bias, can skew outcomes. Moreover, the long-term effects and safety profiles of these interventions are not well-documented, and significant translational gaps remain between research evidence and practical clinical recommendations.
Conclusion
The gut microbiome, a complex network of microorganisms, plays a crucial role in various physiological processes, including nutrient metabolism and immune system regulation. Prebiotics are indigestible food components that stimulate the growth and activity of beneficial microbes in the gut. Through fermentation, prebiotics produce short-chain fatty acids (SCFAs), which confer anti-inflammatory properties and support gut barrier integrity. By promoting the proliferation of beneficial microbes, prebiotics contribute to a healthier gut ecosystem and may protect against gastrointestinal disorders. When consumed in sufficient quantities, probiotics—live beneficial bacteria—can enhance gut barrier function, produce antimicrobial compounds, and modulate immune responses. Probiotics have been shown to alleviate intestinal disorders such as irritable bowel syndrome and antibiotic-associated diarrhea, as well as improve immune function and reduce the risk of infections. Thus, both prebiotics and probiotics play significant roles in improving quality of life by enhancing overall health.
