Food production systems and consumption patterns can have significant consequences for population health, climate, and the environment. Climate change, food insecurity, and an increase in the prevalence of diseases can be associated with unhealthy dietary patterns worldwide, as well as unsustainable food systems (Willett et al., 2019). Current food production systems and agrifood practices have been identified as a major contributor to greenhouse gas emissions and biodiversity loss (Carey et al., 2023). Global meat consumption has risen significantly over the past decade, driven by population growth and increasing incomes (Godfray et al., 2018). Meat is a nutritionally dense food, providing all essential amino acids, high-quality protein, and micronutrients such as zinc, haem-iron, and vitamin B12 (Pereira and Vicente, 2013). However, livestock production systems have been associated with substantial environmental burdens, including greenhouse gas emissions, arable land use, water consumption, and soil degradation (Godfray et al., 2018). Additionally, despite its nutritional benefits, high consumption of red meat has been linked to adverse health outcomes, including cardiovascular diseases (CVDs), colorectal cancer (CRC), and metabolic disorders (MDs) (Pereira and Vicente, 2013). A global modeling study by Springmann et al. (2018) across 150 countries found that reducing animal-product consumption and moving to more sustainable diets in high- and middle-income countries could decrease mortality rates, mitigate environmental impacts from water and land use, and improve public health outcomes. On the other hand, there is some evidence from other database studies that indicates that a drastic shift to environmentally sustainable diets can cause deficiencies in micronutrients, which can especially cause issues for vulnerable members of a population like pregnant individuals and infants (Leonard et al., 2024; Leonard et al., 2023).

Plant-based diets have gained momentum worldwide in the last decade, driven by health, environmental, and ethical considerations (Graça et al., 2015). Figure 1 uses data from Google Trends to illustrate the peak in public interest in plant-based diets and to a lesser extent plant-based protein, before a subsequent dip and slow down (Google, 2025). Consumer surveys in European countries showed a resistance in traditionally omnivorous individuals toward adopting more plant-based foods, with major barriers being nutrition and sensory concerns (Perez-Cueto et al., 2022). The United Nations established the 17 Sustainable Development Goals, for which UN member states have targets of reducing greenhouse gas emissions by 43% by 2030 and achieving net zero by 2050 (Kraak and Aschemann-Witzel, 2024).

Plant protein sources such as legumes, nuts, and seeds contain adequate levels of protein, but are limited by certain intrinsic factors such as cell structure and anti-nutritional factors that impact the nutritional value (Bessada et al., 2019). Plant proteins in their native form typically have reduced nutrient bioavailability than animal-based protein, demonstrating reduced anabolic muscle development, lower levels of essential amino acids leucine, lysine, and methionine, and poorer digestibility due to their protein structure compared to animal-sourced proteins like eggs, dairy, and meat (van Vliet et al., 2015). Most legumes are limited in the amino acid methionine, while grain-derived proteins are limited in lysine. Soy is an exception, as it does contain all the amino acids, however its methionine content may not be adequate for all diets (Gorissen et al., 2018; Shrestha et al., 2023). Contributing to the lower bioavailability are complex plant cell structures and the presence of anti-nutritional factors (ANFs) like trypsin inhibitors, chymotrypsin inhibitors, tannins, and phytic acid (Salim et al., 2023) that can negatively affect the processing and absorption of essential nutrients, including proteins. Plant-based proteins contain a higher proportion of β-sheet and lower α-helix structures compared to animal proteins. α-helices are more water soluble and have accessible structures, meaning they can be broken down by digestive enzymes more easily. The ANF class of trypsin inhibitors in plant material can further reduce plant protein breakdown in the upper gastrointestinal (GI) tract (Bessada et al., 2019).

Proceeding initial digestion in the stomach and small intestine, digested material enters the large intestine containing a very diverse consortia of microbial life. The gut microbiome has been documented as an essential component of human health (Piccioni et al., 2022), so much so that it has been proposed to be considered as a virtual endocrine organ affecting the function of distal organs and systems (Clarke et al., 2014; O'Hara and Shanahan, 2006). While there have been many studies and reviews focused on the role of the microbiome on saccharolytic degradation of resistant starch (RS) or fibers in the colon resulting in the generation of health-promoting metabolites (Adam et al., 2016), the degradation of undigested proteins or resistant protein (RP) by the colon microbiota, known as putrefaction, has received less attention (Wu et al., 2022). Putrefaction can lead to the production of harmful metabolites associated with a range of adverse health effects, including inflammation, intestinal barrier dysfunction, and an increased risk of chronic diseases such as colorectal cancer (CRC) and ulcerative colitis (UC) (Diether and Willing, 2019; Windey et al., 2012). In a traditional balanced diet, putrefaction is usually not an issue as meat, fish, poultry, dairy, and plant proteins diversify daily protein intake with regards to digestibility, secondary nutrients, and amino acids (Yao et al., 2016; Cummings and Macfarlane, 1991), although excessive levels of red meat consumption have been associated with an increase in putrefaction metabolites (Bingham et al., 1996). However, to meet protein demands in a plant-based diet with no animal products, more undigested plant protein would pass through the digestive system than in a balanced diverse diet. There is some evidence to suggest that plant-based proteins in particular are prone to putrefaction, creating issues for the sustainable, widespread adoption of diets containing less meat (An et al., 2014). Indeed, some studies have shown increased populations of putrefying bacteria in the colons of CRC patients compared to healthy adults, which indicates a potential correlation between inflammatory conditions and putrefaction (Wang et al., 2011). However, there is a lack of information on the in vivo mechanisms of putrefaction. This makes it difficult to see a direct association between gut dysbiosis resulting from the accumulation of putrefactive microbes, and human health. Furthermore, identifying mitigation strategies based on clear evidence is challenging.

There is a burgeoning interest in utilizing microbial fermentation as a method in food formulation to improve the digestibility of plant proteins. Fermentation as a food processing tool has been used for centuries across various traditions as a form of preservation. For animal products, especially dairy products such as cheese, yoghurt, and kefir, there have been many studies demonstrating the proteolytic capabilities of fermentation and subsequent improvement in digestibility (Lucia et al., 2014; Fox and McSweeney, 1996; Ferreira et al., 2010). Proteolytic enzyme treatments have also been proposed as a solution to improve digestibility of proteins, as well as host supplementation with probiotics (Goodarzi Boroojeni et al., 2017; Cerdá-Bernad et al., 2022). These proven properties of fermentation could thereby reduce the negative effects of putrefaction in the lower gut. The digestibility of plant protein was shown to have increased in vitro as a result of pre-processing of the plant-based protein via fermentation, hypothesized to be due to enzymatic degradation of ANFs, proteolytic activity, and modification of protein structure, which would all allow proteins to be hydrolyzed to a greater extent in the upper gut (Zhao et al., 2019; Diether and Willing, 2019; Peled and Livney, 2021).

This narrative review examines the potential of fermentation strategies as a processing tool to enhance the nutritional qualities of plant proteins, in turn alleviating some of the negative effects associated with putrefaction. Specifically, it will examine the mechanisms by which putrefaction produces toxic compounds having a subsequent effect on human health, how fermentation modifies plant protein structures and enhances digestibility, and highlights gaps in the current knowledge, identifying pertinent future research directions for the field. The review presents a potential solution in fermentation (see Figure 2) to the less-understood issue of protein putrefaction in the colon, the current state of research of both, and how they might interlink, and future perspectives on understanding the specific mechanisms of their interactions.

While this review is narrative, seeking to highlight a potential gap in knowledge in the field of plant-based nutrition, systematic review approaches have been utilized. Searches were conducted to identify research from two broad areas: (1) protein putrefaction, or (2) colonic fermentation of protein and fermentation of plant protein. The aim of presenting the searches as they were used exactly, is to improve reproducibility of this methodology as this burgeoning field progresses.

According to the World Health Organisation (WHO) (World Health Organisation, 2007), an average adult human should consume 10–15% of their daily calories in the form of protein, representing about 0.83 grams of protein per kilogram of body weight. Protein is an incredibly important component of the diet, needed for normal bodily function, cell repair, and growth (World Health Organisation, 2007). However, the digestibility of proteins in the upper gut or stomach can vary significantly due to a range of intrinsic and extrinsic factors including the structure or three-dimensional folding of the protein, the composition of amino acids, interaction with other components (such as ANFs), post-translational modifications, and polypeptide charges (Gorissen et al., 2018; van Vliet et al., 2015). These variations in digestibility can be a result of the source of protein, how it is processed, other dietary components, and host health.

Initial digestion of proteins occurs in the stomach and small intestine, mediated by pepsin, trypsin, and chymotrypsin. Some undigested proteins and peptides entering the large intestine are further digested by pancreatic enzymes, and the metabolic products are absorbed by the gut microbiota to provide energy and components for cell proliferation via protein production. The human gut microbiome is incredibly diverse, with taxa capable of protein putrefaction identified including Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, and Propionibacterium (Kaur et al., 2017). Putrefaction can result in the release of a plethora of potentially harmful metabolites including nitrogenous compounds such as ammonia (NH₃), N-nitrosamines, amines, phenols, cresols, and indoles (Yao et al., 2016; Korpela, 2018). The effects of these metabolites on health will be explored in a later section. Putrefaction can also result in products associated with positive health effects such as short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) via reductive deamination, or the removal of an amino group. Certain SCFAs, such as butyrate, have been shown to be an important energy source for colonocytes. Butyrate is also implicated in the inhibition of CRC and inflammation (Musch et al., 2001). While SCFAs are also produced via the degradation of fibers and starches, BCFAs are exclusively produced from the deamination of the branched-chain amino acids valine, leucine, and isoleucine, resulting in isobutyrate, isovalerate, and 2-methylbutyrate, respectively. The role of BCFAs was poorly understood, but there is now clinical research showing their involvement in signaling, intestinal maintenance in neonates, and general microbiome health (Lu et al., 2024).

Due to the limitations of studying real-time gut activity, human studies are sparse. However, animal studies, particularly those using pigs as a model of human gastrointestinal activities, are valuable. These studies often look at concentrations of specific toxic compounds remaining in the feces, urine, or present in the gut. The concentration of these compounds often determines their toxicity to the host organisms, which is inversely related to the ability of the colon to absorb and detoxify the compounds. There are potential thresholds below which these compounds would not necessarily be deleterious to health in otherwise healthy subjects, as they would be inactivated by the colon. This is based on the understanding of colon and liver function, depending on the on their ability to detoxify these compounds, which can vary from individual to individual (Yao et al., 2016). However, there is a lack of clarity on the effective toxicity of these compounds in humans, current understanding is based on animal and in vivo studies and these thresholds are not well established (Windey et al., 2012).

As previously described, the composition and diversity of the human gut microbiome influence the degree to which protein putrefaction occurs. In addition, consumption of different types of protein and their level of processing prior to consumption, whether by heat treatment, fermentation, or enzymatic processes, can influence this composition and diversity, with different gut bacteria of varying levels of pathogenicity (Yao et al., 2016). In silico analysis of bacteria based on the putrefaction pathways of all major colonic fermentation products demonstrated that the genus Fusobacterium was abundant in most of the CRC-related datasets compared to healthy adult datasets (Kaur et al., 2017). It was identified as being a risk factor for carcinoma progression in the colon and contains genes coding for a high number of putrefaction pathways associated with toxicity. While all analyzed pathogenic bacteria were found to have some putrefaction pathways, Streptococcus, Candidatus, Escherichia, Shigella, Prevotella, and Selenomonas were found to be in particularly higher levels in patients with CRC. Some commensal gut bacteria were also seen to have putrefaction pathways. Putrefaction by those bacteria may contribute to positive gut health with low levels of putrescine or indole as discussed previously (Kaur et al., 2017).

High levels of animal protein in a diet are associated with a reduction in saccharolytic members of the phylum Firmicutes. These microbes are normally associated with SCFA and BCFA production, reducing inflammatory factors in the gut. The lack of propionate and butyrate leads to a favorable environment for growth of pathogenic bacteria, including Escherichia and Enterococcus (Stecher and Hardt, 2011). The increase in non-digestible oligosaccharides in the form of soy meal in weaning piglets led to an increase in Bifidobacterium (Le Leu et al., 2007). Soybean is a source of complete proteins; it contains all essential amino acids. When fed to rats, it also demonstrated a potential prebiotic effect with increases in lactobacilli and bifidobacteria with a decrease in pathogenic bacteria (Bai et al., 2016). Despite these seemingly positive signs, the higher bioavailability of animal protein such as casein or white meat seems to lead to higher digestibility and less putrefactive metabolite production than soy protein. A lower abundance of Fusobacteria was found in rats fed casein, egg, and white meat protein as compared to plant protein, and both dairy and meat protein fed to rats at recommended levels promoted growth of members of Lactobacillaceae, and Bifidobacterium, compared to soy at similar levels (An et al., 2014). Zhu et al. (2025) compared in vitro fermentation of sheep whey protein versus soy protein isolate using fecal microbiome from healthy volunteers. The authors suggest that the increase in acetic acid in sheep whey protein is due to potential commensal strains that enter the glycolytic pathway. Sheep whey protein also showed higher SCFA, butyric, isobutyric, and isovaleric acids compared to soy protein isolate (Zhu et al., 2025). Despite the presence of RS, the high level of undigested protein in a meal primarily consisting of soy protein leads to putrefaction. Soybeans also contain a large amount of trypsin inhibitors, ranging from 16 to 27 mg/g in raw soybeans (Vagadia et al., 2017), so without removal or reduction of ANFs, this can lead to poor digestion and absorption.

Protein structures and chemical composition can be modified by different processing tools, whether to make protein less or more easily digestible. Non-thermal methods like germination (Jawanda and Ramaswamy, 2024), sonication, pulsed electric field, and high pressure can potentially reduce prevalence of ANFs and cause protein structures to unfold, improving digestibility (Kamiloglu et al., 2024). Similarly, thermal processes like cooking, extrusion, autoclaving, drying, and freezing can break down ANFs and unfold complex protein structures (Skalickova et al., 2022; Purwandari et al., 2024).

Plant protein digestibility can be increased through these mild non-thermal methods that unfold the protein, exposing hydrolysis sites and reducing ANFs. There is an option of utilizing these tools in tandem with fermentation to drive further protein processing (Goodarzi Boroojeni et al., 2017; Jawanda and Ramaswamy, 2024), or allowing access to reaction sites otherwise unreachable by microbes. There are a wide variety of plant protein sources with distinct protein and chemical structures that can be affected by these methods in a variety of ways, so it is important to be specific and consider the fact that these independent proteins are often not the only factor in a diet which can affect digestibility.

As described previously, fermentation is a traditional food processing technology predating domesticated agriculture. Traditionally, it was a form of extending the shelf life of foods prior to the widespread availability of cold storage (Ross et al., 2002). Now, while it is still utilized for preservation, it is also employed as a biotransformation tool to improve sensory characteristics and nutritional value, with the latter being a topic of interest in recent research. Some plant fermentations like the fermented cabbage kimchi (Kim et al., 2023), fermented whole soybean cake tempeh (Ahnan-Winarno et al., 2021), or fermented tea kombucha (Değirmencioğlu et al., 2021) all demonstrate added value through improved antioxidant content, digestibility, and bioaccessibility. However, there has only been recent renewed research interest in plant protein fermentation, with the intention of creating a highly digestible fermented product with comparable protein quality to animal protein. Heat-labile ANFs like trypsin inhibitors and lectins (as discussed previously) are readily reduced through high thermal or extrusion cooking in conventional plant protein processing. However, heat-stable ANFs, like phytic acids and phenolic compounds, remain in the product, worsening digestibility and potentially leading to putrefaction. Some studies involving fermentation of plant protein will be explored, along with their effects on overall nutrition.

As such, Table 1 depicts a variety of examples of experimental studies exploring the impact of fermentation of plant protein directly on protein digestibility. The limitations of some of this data are that there is a lack of replication of their work, as well as a lack of controls or sterilization of substrates used that could confound collective conclusions. Furthermore, many of these studies also look at ‘natural’ or spontaneous fermentations, which are challenging to experimentally replicate.

Despite the review’s focus on fermentation, studies emphasize resistant starch (RS) for gut health, as TIM-2 models show RS inclusion prevents putrefaction by boosting saccharolytic fermentation (linked to SCFAs) and commensals like Phocaeicola vulgatus. Branched pectins, fermented in the distal end of the colon where putrefaction occurs, prioritize this beneficial process, countering proteolytic toxicity (Toden et al., 2006).

There are several vitamins, minerals, and other metabolites that humans do not endogenously produce and hence require external sources for them. Certain commensal members of the gut microbiome, especially certain strains of the genus Bifidobacterium, have been demonstrated to convert dietary components into bioactive molecules including vitamins. Several Bifidobacterium strains produce B-group vitamins, e.g., folate at high levels (Bifidobacterium bifidum) or low levels (Bifidobacterium breve), confirmed by increased fecal levels of the vitamin in animal and human studies. Lactic acid bacteria found commensally can also contribute to folate production. Some strains from the species Limosilactobacillus reuteri found in fermented foods were shown to produce corrinoids like vitamin B₁₂ which is not found in adequate quantities in a typical plant-based diet. There have been other commensal species that have been shown in vitro to produce other important compounds like vitamin K, nicotinic acid, and secondary bile acids. Fermented foods can be rich in SCFAs, BCFAs, and free metabolites that could potentially drive growth of saccharolytic bacteria.

Looking at mechanistic evidence of how RS is processed in health-promoting fermentation pathways can inform on what members of the gut microbiome are responsible, and what specific compounds cause positive health effects. While fermentation does not add resistant starch to foods, this could also potentially inform ways of carrying out pre-fermentation of plant-based foods to maximize their nutritional value (He et al., 2017; LeBlanc et al., 2013). By selecting strains that can add those positive compounds or targeting them, and further studying specialized fermentations can be prioritized.

There are two points of interest that must be addressed to clearly indicate whether fermentation would be useful as a tool to prevent putrefaction of plant protein. Firstly, the mechanisms of putrefaction of plant protein in humans must be understood, as well as whether putrefactive compounds are truly associated with deleterious health conditions. Secondly, the interactions between unfermented proteins or fermented proteins and putrefactive bacteria must be compared after gastrointestinal digestion.

To answer the first question, double-blind human intervention studies may prove valuable in seeing changes in the gut microbiome on consumption of a high-plant-protein diet versus a normal omnivore and high-animal-protein diet. While this would not be perfect, as gut microbiome changes may occur over a significantly longer period, any changes in putrefactive bacteria may allow initial conclusions to be drawn. This, paired with cell signaling and molecular experimentation of the effects of putrefaction compounds on human cell lines, can help provide more understanding. Further animal trials with transgenic pigs or mice may elucidate these potential systems in a live body.

To connect the effects of fermented plant protein with putrefaction, a protocol similar to the first proposed experiment could be followed. Fermented plant proteins that show a high degree of protein digestibility, both in vivo and in vitro, can be chosen and used in an intervention study like the one previously described. Depending on the presence or absence of putrefactive bacteria in the fecal microbiome of participants fed fermented samples versus non-fermented, inferences can be drawn on the effectiveness of fermentation as a mitigation tool. Furthermore, GI disease animal models can be utilized and addition of fermented foods in feed can be used to determine if any change in gut microbiome or disease markers has been observed. Traditional fermented foods must also be examined in more controlled environments, as points of references for future bio-processing methodologies. While microbial consortia may vary greatly in spontaneous fermentations, large sample sizes may help to identify trends in microbe type, protein modifications, metabolites, and effects on cells and gut microbiota. There is some evidence to suggest that prolonged fermentation times may result in reduced amino acid scores due to microbes metabolizing certain amino acids (Çabuk et al., 2018), so more work must also be done in optimizing fermentation conditions as well.

As discussed in this narrative review, putrefaction is a complex process that produces harmful metabolites that have been implicated in inflammatory and chronic diseases of the gut. The shift toward plant-based diets, driven by a multitude of concerns, has highlighted the need to address the challenges associated with plant protein digestibility and the link between this and putrefaction. Plant-based proteins, while potentially environmentally sustainable, high in fiber, and rich in essential nutrients, often suffer from lower bioavailability, structural complexity, and the presence of ANFs. For these proteins to be a completely sustainable alternative, they must be capable of sustaining human nutritional needs. Fermentation offers a potential solution; by degrading ANFs, modifying protein structures, and breaking down complex proteins, fermentation can improve the digestibility and nutritional quality of plant proteins, reducing their potential for harmful putrefaction. For example, fermentation of legume proteins by LAB and fungi has been shown to reduce ANFs, increase amino acid bioavailability, and improve protein digestibility in vitro. The topic of putrefaction as demonstrated is a complex one; it results in the release of metabolites in the gut, and some of those metabolites have been shown to cause potential harm in controlled laboratory or animal models. However, currently, the evidence linking poor digestion of protein to CRC, UC, and other gastrointestinal disorders is not strong enough to be conclusive. There is currently a lack of experimental evidence showing direct links between fermented food consumption and protein putrefaction mitigation in humans, or even a definitive case for putrefactive compounds consistently causing gastrointestinal disease. Most studies to date have been primarily conducted using in vitro digestibility or using in vivo animal models (rats, weaning pigs), limiting their direct applicability to humans. Furthermore, the variability in plant protein sources, processing methods, and microbial consortia used in fermentation necessitates more standardized and controlled human trials to validate these findings. The degree of fermentation, and ways to utilize it as a bio-processing tool must be identified, to reach a conclusion on optimal fermentation conditions.