Understanding the nutritional benefits through plant proteins-probiotics interactions: mechanisms, challenges, and perspectives

Abstract

The nutritional benefits of combining probiotics with plant proteins have sparked increasing research interest and drawn significant attention. The interactions between plant proteins and probiotics demonstrate substantial potential for enhancing the functionality of plant proteins. Fermented plant protein foods offer a unique blend of bioactive components and beneficial microorganisms that can enhance gut health and combat chronic diseases. Utilizing various probiotic strains and plant protein sources opens doors to develop innovative probiotic products with enhanced functionalities. Nonetheless, the mechanisms and synergistic effects of these interactions remain not fully understood. This review aims to delve into the roles of promoting health through the intricate interplay of plant proteins and probiotics. The regulatory mechanisms have been elucidated to showcase the synergistic effects, accompanied by a discussion on the challenges and future research prospects. It is essential to recognize that the interactions between plant proteins and probiotics encompass multiple mechanisms, highlighting the need for further research to address challenges in achieving a comprehensive understanding of these mechanisms and their associated health benefits.

Keywords:

• Probiotics
• plant protein
• synergistic effect
• metabolism
• interaction

1. Introduction

Plant-based proteins foods, characterized by low fat and calorie content, lactose-free composition, and other beneficial traits, are gaining popularity as a viable alternative for individuals seeking to replace animal proteins. Particularly, this trend resonates with vegetarians and lactose-intolerant individuals, affirming their status as rising stars in the plant nutrition market (Aimutis 2022; Yan et al. 2023). The range of available plant proteins has been steadily expanding, encompassing various sources such as cereals (oats, rice), pseudocereals (buckwheat, quinoa), legumes (soybeans, peas, mung beans, chickpeas), and nuts (almonds, walnuts). This market expansion has garnered significant interest from the industry (Hertzler et al. 2020).

The word "probiotic" comes from the Greek meaning "for life". Probiotics are a group of living microorganisms that play a pivotal role in the fermentation of diverse food items, spanning from vegetables and fruits to meats, dairy, and grain products. Their application not only elevates the nutritional value of these foods but also offers numerous health benefits when consumed in adequate doses (Hill et al. 2014). In recent years, due to the increasing demand for preventive health care by groups worldwide, the market size of probiotics has grown strongly and is expected to grow to $119.28 billion by 2028 (Intelligence 2024). Commonly found in fermented foods or consumed as dietary supplements, probiotics are a valuable addition to any balanced diet. The incorporation of probiotics fermentation in food preparation can alter the taste and composition of the final products. Specifically, the application of probiotics fermentation to different plant proteins can effectively break down anti-nutritional factors, thereby enhancing the overall nutritional value of the food. Additionally, probiotics have the ability to improve the flavor and texture of plant-based beverages, allowing for the mitigation of undesirable taste and texture characteristics. Compared to conventional vegetable protein drinks, probiotic-fermented plant-based protein beverages offer distinct advantages in terms of vitamin content, improved nutrient absorption and bioactivity, product stability, as well as safety considerations (Valero-Cases et al. 2020).

Probiotics are able to effectively regulate pH levels to improve the solubility and digestibility of plant proteins. Through fermentation, probiotics can modify the structure and functionality of plant proteins, resulting in the production of bioactive substances such as vitamins, antioxidants, and antimicrobial peptides. Furthermore, probiotics facilitate protein absorption and utilization, thereby addressing protein energy malnutrition. Additionally, probiotics alter the metabolic activity within the intestinal microbiota, maintaining a balance between protein degradation and synthesis (Rasika et al. 2021).

The combination of plant proteins and probiotics exhibits synergistic effects, whereby plant proteins create a favorable environment for probiotics growth and proliferation. Serving as a substrate for probiotic metabolism, plant proteins support their growth and contribute to the synthesis of beneficial metabolites. This reciprocal interaction between plant proteins and probiotics enhances their overall effectiveness in promoting gut health and facilitating nutrient absorption. The combination of protein and probiotics holds significant potential for promoting human health, with benefits including improving digestion, maintaining gut health, supporting for the immune system, enhancing mental well-being, and improving nutrient absorption (Ranjha et al. 2021).

The combination of multiple flavors, nutrition, food functionality, process refinement, and probiotics with plant proteins has become a focal point of current research. The fusion of plant proteins and probiotics can cater to various nutritional needs for human health while also delivering innovative taste and nutrition. These synergistic effects show significant potential in enhancing protein digestion and absorption thereby improving human health. The combination of specific probiotic strains and plant proteins is paving the way for advancing plant protein products. However, the mechanisms and challenges of these synergistic effects for health promotion necessitate further investigation. This review seeks to delve into the roles of promoting health through the interplay of plant proteins and probiotics. The regulatory mechanisms have been elucidated to demonstrate the synergistic effects, with a discussion on the challenges and prospects for future research.

2. The interactions between plant proteins and probiotics

Plant protein plays a crucial role in regulating the growth and metabolism of probiotics. Previous studies have shown that protein stimulates the synthesis of cytoplasmic proteins in probiotics, which in turn increases cell biomass and growth rate (Molska et al. 2022). Additionally, through fermentation (Figure 1), probiotics can alter the structure, modify functionality, facilitate bioactivity availability of plant proteins, regulate the absorption, and utilization of nutrients. Plant proteins are instrumental in improving the growth and proliferation of probiotics, stimulating the synthesis of cytoplasmic proteins in probiotics, supporting energy and metabolism, as well as maintaining the integrity of cell membranes. The combination of protein and probiotics demonstrates significant potential in promoting human health, including maintaining gut barrier, altering the metabolic activity within the gut microbiota, improving gut homeostasis, and enhancing immune response. By integrating plant proteins with probiotics, it becomes possible to meet diverse nutritional requirements while simultaneously achieving innovative taste and nutrition, thus offering promising applications.

Figure 1. The interactions between plant proteins and probiotics regulate nutrients metabolism and probiotic growth and proliferation.

2.1. The promotion of probiotics growth and metabolism

Probiotics need a supply of nutrients for proliferation, and plant protein act as an essential growth stimulant for probiotics (Shaiken et al. 2023). It has demonstrated that protein promotes the synthesis of probiotic cytoplasmic proteins, thereby increasing the cell biomass and growth rate (Molska et al. 2022). Amino acids and peptides in protein degradation products can be converted into energy and metabolites through metabolic pathways. The energy generated by this process can be used to maintain the basic metabolic needs of cells, as well as maintaining the integrity and stability of cell membranes (Canon et al. 2021). The amino acids sequence in protein can affect the activity of the enzyme inside the probiotics. The types and contents of amino acids in proteins have significant effects on the growth rate and biomass of probiotics (Wang, Rosa-Sibakov, et al. 2022).

In addition, probiotics can use protein degradation products as carbon and nitrogen sources for growth and metabolism. Carbon and nitrogen sources have important effects on the growth and metabolism of probiotics (Xie et al. 2023). Protein can serve as carbon source to provide the necessary carbon elements for probiotics growth and metabolic activities (Bamigbade et al. 2022). At the same time, protein can also be used as a nitrogen source, in which amino acids can provide nitrogen elements required by probiotics to synthesize proteins such as cell structure and enzymes (Plavec and Berlec 2019). Moreover, the peptides and oligopeptides can be produced by protein hydrolysis or self-hydrolysis are deemed to regulate the activity and stability of enzymes and thus promote the growth of probiotics (Ramírez, Pineda-Hidalgo, and Rochín-Medina 2020). After the combination of probiotics and plant proteins, probiotics have a better survival chance, at the same time, play the physiological activity of probiotics and the growth-promoting function of prebiotics to produce health benefits to the host (Liu, Wang, and Wu 2021).

The effects of different types of proteins on probiotics are also different to some extent. Ding and Li (2021) found that when walnut oligopeptide was added at 2.0%, the number of viable bacteria reached 2.69 × 10¹⁰ CFU/mL, which increased the metabolic rate, promoted the proliferation of probiotics, and improved the activity of bacteria. Zhang et al. (2020) found that the peptides with small molecular weight from both soybean protein isolates and soybean peptides (PEPs) can be easier to effectively promote the growth of L. rhamnosus. It was also reported that the role of the soybean oligopeptide on JCM 1132 strain was manifested as promoting bacterial growth by increasing the content of tricarboxylic acid circulating intermediates, improving the metabolic capacity of L. acidophilus JCM 1132 (Li et al. 2020). In vitro simulated digestion tests confirmed that PEPs can act as a nitrogen source to promote probiotics growth and produce more lactic acid and acetic acid. These acids are derivatives of glucose metabolism by probiotics, demonstrating their efficacy in suppressing detrimental microorganisms (Chi et al. 2021).

Probiotics fermentation also produces metabolites with antibacterial properties, such as organic acids, peptides. Studies have shown that after fermentation of soy milk with probiotic B. coagulans VHProbi C08, L. rhamnosus and L. helveticus K14, it has considerable antimicrobial capacity against Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, Bacillus cereus and Staphylococcus aureus (Peng et al. 2021). The metabolites produced after the combination of proteins and probiotics can affect the growth and proliferation of probiotics and further promote the bioactive function of probiotics (Leeuwendaal et al. 2022). Table 1 has listed the recent studies to verify the influence of different plant proteins on probiotics growth and metabolites, promote the proliferation of probiotics. However, the specific effects can differ according to the probiotics type and multifactorial environmental. Further studies are required to fully understand how different plant proteins interact with probiotics, as well as their impact on gut health.

Table 1. The influence of different types of plant proteins on probiotics proliferation.

Probiotics strain	Plant protein	Fermentation condition	Experimental method	Performance	Reference
B. animalis subsp. animalis JCM 1190	Soybean	With 5% inoculum and anaerobic culture at 37 °C for 48 h.	The spread plate method	The growth ability of Ba1190 enhanced, the viable cell number reached the highest after 24 h: Pro (8.6 × 10^8 CFU/mL), dPro (9.7 × 10^8 CFU/mL).	(Li et al. 2021)
L. plantarum 45	Soy	Inoculate 1% into MRS-protein medium (0.5 or 1 mg/mL SP). Incubated at 37 °C for 24 h.	Enzyme marker measured growth curve, and dilution coating method	1 mg/mL SP group had higher total number of colonies (6.5 × 10^9 CFU/mL) and the growth period of LP45 shortened.	(Liu et al., 2022)
L. plantarum Z7	Walnut	0.5%, 1.0%, 1.5%, 2.0% WOPs, and 1% L. plantarum. Incubated at 37 ^oC, 160 rpm for 48 h.	Microbial growth dynamics model (Modified Gompertz model and Logistic model)	The viable bacteria count reached 2.69 × 10^10 CFU/mL, the metabolic rate and activity increased, and promoted the proliferation.	(Ding and Li 2021)
L. acidophilus JCM 1132	Soybean	Cultured at 37 °C for 48 h with 2% (v/v) L. acidophilus.	Dilution plate counting method	The growth and metabolism capacity of L. acidophilus was improved with a cell count of 42.0 × 10^7 CFU/mL.	(Li et al. 2020)
L. rhamnosus	Soybean	The nitrogen contents of the SPIs, PEPs, dSPIs and dPEPs were 134.7, 143.58, 109.3 and 101.4 mg N/g. 6 × 10^7 CFU L. rhamnosus. Sampled at 37 °C every 4 h for 16 h.	A microplate reader measured cellular growth, and plate colony counting method	The growth curve showed an increasing trend, lower OD values with 5.6 × 10^8 CFU/mL viable bacteria count.	(Zhang et al. 2020)
L. rhamnosus Lra05	Soy	200 μL L. rhamnosus was transferred into 50 mL medium. Cultured for 24 h at 37 ^oC.	Dilution plate counting method	The number of viable cells in M group was lower than dpep and dpro (4.03 × 10^9 and 3.55 × 10^9 CFU/mL).	(Chi et al. 2021)
S. thermophilus LMD-9	Soya	By inoculating milk with the frozen stock culture (1 × 10^6 CFU/mL) and incubation at 37 ^oC.	Enumeration and semi-logarithmic growth curves	Acidification occurred in tandem with increasing population size (10^9 CFU/mL). S. thermophilus can grow by consuming sucrose.	(Boulay, Al Haddad, and Rul 2020)
L. plantarum 299v	Quinoa	Quinoa flour and demineralized water (1:2 w/v). Incubated at 30 °C for 10 h with 7.35 Log^®10 CFU/g L. plantarum.	Plate viable colonies count	The cell growth in processes 1, 2b (10.0 ± 0.14), and 2c (10.1 ± 0.21) was higher than 3b (8.29 ± 0.07) due to fermentation.	(Castro-Alba et al. 2019)
L. casei 0979	Red clover seed, chickpea, lentil and alfalfa	50 g sprouts with 50 mL inoculum suspension. Fermentation at 30 °C for 48–96 h with 10^7-10^8 CFU/mL.	Plate counting method	LAB increased by 2 LU. E. coli and Klebsiella sp. reduced.	(Budryn et al. 2019)
P. pentosaceus, P. acidilactici	Chickpea	300 μL LAB suspensions, 20 g sterilized demineralized water, 40 g chickpea flour. Incubated at 37 °C for 72 h.	Incubate the colonies were counted	LAB increased by 0.4-1.4 log CFU/g. Compared with P.a.5 strain (7.4 log CFU/g), non-fermented had lower LAB (6.0 log CFU/g).	(Xing et al. 2019)
L. paracasei I1, L. plantarum M4, L. brevis T4	Quinoa and wheat	100% wheat/quinoa/Kamut^® flour or 25% wheat and 25% quinoa and 50% Kamut^® flour with starter (∼7, 7.5 log CFU/g). Incubated at 48 h for 30 ^oC.	Counting by dilution and flat plate	After 24 h, with counts higher than 9.0 log CFU/g. L. paracasei was better adapted to quinoa matrix (10.0 log CFU/g).	(Di Renzo et al. 2018)
L. acidophilus NCFM^®, S. thermophilus, L. delbrueckii subsp. bulgaricus, B. lactis HN019^™	Pea	4% protein solution with 10^7 CFU/mL VEGE and T. delbrueckii. Fermentations were stopped at pH 4.55.	Counted by spread plating technique	VEGE + K. lactis presented higher bacteria (6.0 ± 1.6 × 10^8 CFU/mL) than VEGE, VEGE + K. marxianus and VEGE + T. delbrueckii.	(El Youssef et al. 2020)
L. plantarum DSM 9843	Quinoa	Mixed quinoa grains with sterile water (1,4 w/v), inoculated with 1.8 × 10^7 CFU/bottle probiotics. Fermented at 30 °C for 2 days.	Pick colonies from the countable plates	The Lactobacillus count was high 10.6 log CFU/mL after 48h, and a significant increase of 9.82 log CFU/mL was shown until day 14.	(Canaviri Paz, Janny, and Håkansson 2020)
L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2	Pea and brown rice	Mixture of pea and rice protein (13%) with 10^8 CFU/mL LAB. Fermented at 37 °C for 14 h.	Spread plate method	PRF exhibited higher Lactobacillus (9.45 log CFU/mL) at end of fermentation.	(Zahra et al. 2022)

Note: L. plantarum, Lactobacillus plantarum; L. rhamnosus, Lactobacillus rhamnosus; L. acidophilus, Lactobacillus acidophilus; L. casei, Lactobacillus casei; L. brevis, Lactobacillus brevis; L. delbrueckii, Lactobacillus delbrueckii; B. animalis, Bifidobacterium animalis; S. thermophilus, Streptococcus thermophilus; E. coli, Escherichia coli; P. pentosaceus, Pediococcus pentosaceus; P. acidilactici, Pediococcus acidilactici; T. delbrueckii, Torulaspora delbrueckii; K. lactis, Kluyveromyces lactis; K. marxianus, Kluyveromyces marxianus.

SP, soybean protein; WOPs, walnut oligopeptides; SPIs, soybean protein isolates; PEPs, soybean peptides; dpro, digested soybean protein; dpep, digested soybean peptides; dSPIs, digested soybean protein isolates; MRS/M, Man Rogosa Sharpe broth; PRF, pea and rice proteins; LAB, lactic acid bacteria.

2.2. The enhancement of plant proteins functionality

2.2.1. The enhancement of plant proteins degradation

Proteolysis is triggered by cell envelope proteinase that breaks down proteins into polypeptides. Subsequently, dipeptides, tripeptides and oligopeptides may be taken up into the cells. In the metabolic pathway involved in metabolites of proteolysis, various enzymes play crucial roles, including cell envelope proteinase, peptidases, biosynthetic enzymes, dehydrogenase, aldolases, lyases, acyltransferases esterases, dehydrogenase, aminotransferases, decarboxylase, deiminases decarboxylase, dehydrogenase complex, and additional biosynthetic enzymes. Probiotics increase the production of protease and the enhanced activity of protease makes free amino acids a necessary factor for the proliferation of probiotics and also produces a variety of bioactive peptides (Brown et al. 2017). Furthermore, probiotics can induce the host to enhance the activity of digestive proteases and peptidases, as well as the release of exoenzymes involved in protein digestion (Figure 2A). Peptidases such as PepN and PepXP play an essential part in releasing amino acids, cell growth, and biomass formation and are also involved in the production of precursors of flavor substances (Wang and Ji 2019).

Figure 2. The roles of probiotics on proteolysis (A); the direct/indirect pathways of probiotics for gut microbiota-mediated proteolysis (B); and the effects of microbial metabolism on nutrient quality factors of plant food matrix (C).

FAA, free amino acids; BCAA, branch-chain amino acids; EAA, essential amino acids; IVPD, in vitro protein digestibility; IL, interleukin.

In the process of fermenting broad bean protein with the combination of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus acidophilus, more active protease will be produced, and plant proteins will be decomposed due to the synergistic effects between strains. After protein degradation and separation, small-molecule active polypeptides will be produced, and thus improve the nutritional quality and itself functional features (Ma et al. 2023). The fermentation uses proteases secreted by probiotics to hydrolyze proteins, optimize the texture, nutritional value, and bioactive substances of each plant source, and further promote the development of healthy fermented plant proteins (Engels et al. 2022).

In the process of fermenting plant proteins, probiotics can help to release and improve the active factors and metabolites and improve the utilization of plant proteins. Meanwhile, the probiotics will be in the lag or decline period and the secondary metabolites generated will decompose the plant proteins into small molecular peptides and free amino acids and then accumulate more amino acids and peptides (Zhao et al. 2022). At the same time, the variations among the strains themselves will lead to diverse metabolism and utilization capacities of plant proteins. This phenomenon may be caused by varying microorganisms producing different enzymes in the growth and reproduction process and different positions or degrees of plant proteins being cut, resulting in large differences in the amino acids composition types generated (Zhou et al. 2022).

2.2.2. The improvement of plant proteins digestion and absorption

Plant proteins digestion plays an important role in body health, as it can release bioactive peptides that are beneficial to overall well-being. Probiotics can influence the processes of proteins breaking down and amino acids synthesis mediated by intestinal bacteria through direct and indirect pathways (Figure 2B). Direct pathways include the enhancement of exogenous digestive enzymes. Indirect effects include modulation of microbial composition to support gut homeostasis, including the intestinal epithelial cells (IECs) normal morphology, thus providing an appropriate microenvironment for dietary plant proteins metabolism.

As shown, probiotics can enhance the digestion and uptake by directly influencing metabolism processes and indirectly manipulating the host gut ecosystem. Through a series of metabolic reaction processes, probiotics make biochemical changes in plant proteins and finally realize the mutual conversion between the three major substances (sugars, lipids, and proteins) or produce new substances to promote the digestion, decomposition and absorption of nutrients. Probiotics fermentation will cause enzymatic hydrolysis and acidification reactions, which will change functional properties of food and affect the advanced structure of proteins (Jiang et al. 2020).

The increase in protein digestibility is related to the decrease in α-helix to β-fold ratio, which produces acetic acid and lactic acid after microbial fermentation, leading to the instability of the protein secondary structure and rearrangement phenomenon (Yasar, Tosun, and Sonmez 2020). It significantly improves the solubility and digestibility of plant proteins and promotes amino acids production and protein degradation, which means that plant proteins treated with probiotics are more easily absorbed and utilized by the human body (García Arteaga et al. 2022).

Bacillus coagulans GBI-30, 608 in combination with plant protein has been proven to enhance the absorption and utilization of the protein and optimize the nutritional value in connection with protein supplementation (Keller et al. 2017). BC30 promotes digestion and uptake of pea, soybean, or rice protein in the upper gastrointestinal tract and maintains a healthy colon microenvironment (Kim et al. 2023; Walden et al. 2022). An in vitro study found that using Bifidobacterium animalis subsp. lactis B420, Lactococcus lactis subsp. lactis Ll-23, Lactiplantibacillus plantarum Lp-115, Lactobacillus acidophilus NCFM, Lacticaseibacillus paracasei subsp. paracasei Lpc-37, B. lactis Bl-04 and Lacticaseibacillus rhamnosus HN001 fermented plant proteins could affect the capacity of digestion while increasing bioavailability and accessibility (Marttinen et al. 2023).

The addition of probiotics can increase the absorption of amino acids and small peptides by plant proteins. The synergistic action of plant proteins with L. paracasei LP-DG^® and LPC-S01 increases the amino acid concentration in blood, such as methionine, histidine, valine, leucine and tyrosine (Jäger et al. 2020). Furthermore, it promotes the utilization of proteins by enhancing the function and activity of intestinal cells, increasing the absorption surface area of IECs, improving the absorption capacity of the epithelium, and promoting peptide transport.

After the probiotics treatment, the plant protein changed from a tight ball structure to a loose wire group structure, and some groups appeared, which had a greater impact on the protein molecules, thus enhancing the absorption effect of plant proteins by the human body (Yakubu et al. 2022). The defect of low digestibility of plant proteins caused by the tight arrangement of protein structures is improved. Fermentation promotes the hydrolysis of proteins and the production of amino acids, which in turn increases the digestibility of plant proteins. However, different probiotics perform different abilities to produce flavor compounds for different plant proteins. Table 2 has highlighted the roles of different probiotic strains and plant proteins, fermentation conditions, in vivo and in vitro experimental models, and specific change indicators to certify promote protein digestion and absorption.

Table 2. The influence of different types of probiotics on plant proteins digestion and absorption.

Probiotics strain	Plant protein	Fermentation condition	Experimental model	Performance	Reference
L. acidophilus, L. plantarum, L. casei, L. rhamnosus, L. mesalococcus, L. lactis, L. paracasei, L. bulgaricus, and S. thermophilus	Soy	10% SPI dispersions with 2% glucose, 4% strains. Incubated statically at 37 ^°C for 6 h.	In vitro gastrointestinal digestion with a two-step protocol	The microstructure of the fermentation-induced gels may influence digestion.	(Yang, Ke, and Li et al. 2021)
L. delbrueckii subsp. bulgaricus and S. thermophilus	Oat	600 g oat protein concentrate (OPC) in 4 kg distilled water with 2.8 g starter. Fermentation at 45 °C for 24 h.	OPC-yoghurt, in vitro	During fermentation, proteolysis occurred and proteolytic enzymes cleaved oat protein and released peptides.	(Brückner-Gühmann et al. 2019)
Not mentioned (mixed strains)	Foxtail millet (Setaria italica L.)	250g foxtail millet seeds,10^4-10^5 CFU/mL bacterial. Fermentation at 38 ^°C for 20 h.	In vitro protein digestibility (IVPD)	Microbe produced proteases and fermentation exhibited higher IVPD than untreated (74.28 vs 68.65 g/100 g).	(Sharma and Sharma 2021)
L. casei IDCC 3451	Pea and soy	Legumin mixture with 18% and 40% protein content, L. casei (5 × 10^8 or 5 × 10^9 CFU/day) was orally administered for 8 wk.	C57BL/6 J mice (6-week-old male), in vivo	It helps to absorb AA by proteolyzing proteins.	(Kim et al. 2023)
B. coagulans GBl-30, 6086 (BC30)	Pea, soy and rice	1 × 10^9 CFU B. coagulans, three plant protein with 90%, 82%, 82.3% protein content.	In vitro model of the stomach and small intestine (TIM-1)	BC30 increased proteins digestion and uptake, shorter peptides and more FAA, showed a 2-fold reduction in TN/AAN ratio.	(Keller et al. 2017)
W. coagulans GBI-30, 6086	Rice and pea	20g protein concentrate with 1 × 10^9 CFU BC30. Two separate 14-day protocols.	Healthy, older females (n = 30), in vivo	BC30 improved CMax. Ala (p = 0.018), Try (p = 0.003), Cys (p = 0.041), EAA (p = 0.050), and AA (p = 0.039) showed greater AUC.	(Walden et al. 2022)
L. paracasei LP-DG^® (CNCM I-1572), LPC-S01 (DSM 26760)	Pea and rice	20 g pea protein with 5 billion CFU LP-DG^® plus 5 billion CFU LPC-S01. 2 wk of trial design.	Clinical Study, healthy men (TCU = 10; UMHB = 5; n = 15), in vivo	The increase of Met (16.3%), His (49.2%), Val (24.7%), Leu (25.2%), Ile (26.1%), Tyr (11.6%), BCAA (26.8%) and EAA (15.6%) CMax.	(Jäger et al. 2020)
L. plantarum CKDHC 0801 and L. brevis KCCM 11509	Pea	11.1% (w/w) IPP solution, starter culture (>3 billion CFU/mL). Fermentation for 1 h at 90 ^oC.	In vitro digestion model.	Enzyme-modified and EFPP resulted in 22.50% more digestion than IPP, and higher bioavailability and 53.92% reduction in β-sheet.	(Hyeon Deok et al. 2023)
B. subtilis spp. natto	Soybeans	150 g boiled soybeans with 50 mL diluted culture from a commercial natto product. Fermentation at 40 °C for 18 h.	Simulated the static in vitro gastrointestinal digestion model	Fermentation improved IVPD by 45% and absorption of oligopeptides. EAA and TCA-soluble protein (SP) content (59.83%; G2I2) were higher.	(Ketnawa and Ogawa 2018)
L. plantarum NRRL B-4496	Pea	Fermentation anaerobically at 32 °C for 11 h with 7 log CFU/g.	In vitro protein digestibility	Protein digestibility reached max (87.4%) after 5 h. The sulfur and IVPD-corrected AAS reduced (from 0.84 to 0.66, 67 to 54.6%, 0–11 h).	(Çabuk et al. 2018)
B. animalis subsp. lactis B420, B. lactis Bl-04, L. acidophilus NCFM, L. rhamnosus HN001, L. paracasei subsp. paracasei Lpc-37, L. plantarum Lp-115, and L. subsp. lactis Ll-23	Soy and pea	Soy and peas provided 90.0% and 83.0% protein. Inoculated at 10^8 bacteria in the oral phase.	In vitro digestion model of the upper GIT	All strains increased FAN (13%-33) or FAA concentration. The SP content was 67% and 52% higher in B420 and Ll-23.	(Marttinen et al. 2023)
L. plantarum B1-6	Soy	Fermented 3.0% (v/v) SPI solution and terminated when pH reached 7.0, 6.0, 5.0, and 4.0.	In vitro dynamic gastrointestinal digestion	The FSPI-7.0 digestate exhibited the peak SP level (8.45 mg/mL, I-5 min), but lowest at I-180 min.	(Yaqiong Wang, Rosa-Sibakov, et al. 2022)
L. plantarum IDCC 3501	Soybean	40% high-protein with 5.0 × 10^9 or 10^8 CFU/day L. plantarum for 8 wk.	C57BL/6J male mice, six weeks, in vivo	Ala, Val, Ile and Leu showed higher concentrations (78.88 ± 77.89, 66.28 ± 34.67, 22.57 ± 14.34 and 50.61 ± 27.27 µmol/L).	(Jeon et al., 2023)
B. subtilis, L. plantarum, and S. cerevisiae	Soy	B. subtilis, L. plantarum, and S. cerevisiae at ≥10^9, ≥10^10, and ≥10^9 CFU/mL.	Crossbred male weaned piglets, in vivo	FSB piglets had higher crude protein ATTD (85.39 ± 0.77). The ether extract ATTD in FSB was higher than MFSM (p < 0.05).	(Li et al. 2022)
L. plantarum ATCC 8014	Peas	10 g pea flour with 500 µL bacterial (1.5 × 10^8 CFU/mL). Fermentation for 72 h at 37 ^oC.	In vitro digestion according to INFOGEST	A significant increase in releasing Phe, Leu, Asn, Asp, Met, Glu, Val and Cys, with higher total proteins.	(Skalickova et al. 2022)
L. paracasei L9, L. bulgaricus and S. thermophilus	Soy	2% starters (about 6.4 log CFU/mL). Fermentation at 37 °C for 24 h then storage (4 °C, 1–21 d).	Soymilk samples, in vitro	Fermentation broke the highly aggregated proteins and product small peptides. The total FAA of L9SM and CSSM increased by 68% and 134%.	(Wu et al. 2022)
L. delbrueckii subsp. bulgaricus and S. thermophillus	desi, kabuli chickpea and faba bean	0.5 g yogurt culture. Fermentation at 30 °C for 16/20 h.	IVPD	Protein contents in fermented faba bean were higher. IVPD was increased (9.5%, desi) and trypsin inhibitor activity reduced by 2.7%.	(Chandra-Hioe, Wong, and Arcot 2016)
L. plantarum VTT E-78076	Faba bean	10^6 CFU/mL cell density. Triplicate fermentations for 24 h at 25 ^oC.	In vitro digestion model	Fermentation caused higher FAN and lower SP content, improving the protein digestibility.	(Rosa-Sibakov et al. 2018)
L. bulgaricus and S. thermophilus	Tigernut	Inoculated with starter culture at 5 g to 1 L. Fermentation at room temperature for 8 h.	Yogurt sample, in vitro	The total amount of NEAA higher than EAA. The tigernut yogurt be abundant in Glu (2.863%-5.746%).	(Ogundipe et al. 2021)
L. rhamnosus SP1, W. confusa DSM 20194, L. plantarum T6B10	Quinoa	Initial concentration ca. 6 log CFU/mL. Fermentation at 30 °C for 20 h and stored at 4 °C for 20 days.	IVPD, a multi-step protocol	NI was the highest in B-SP1 (5.8 ± 0.2, T20). IVPD and protein score increased to 80–86% and 37%. EAAI, PER, and BV increased up to 20% (T0-Tf).	(Lorusso et al. 2018)
L. acidophilus CL1285, L. casei LBC80R, L. rhamnosus CLR2	Pea, rice and hemp	50/50 blend of pea and brown rice protein (PR), or 30/70 blend of pea and hemp protein (PH). Fermentation at 37 ± 1 °C for 14 ± 2 h with 10^8 CFU/mL.	In vitro digestibility procedure, pepsin and trypsin digestion model	The fermentation of PR increased (61.4% to 72.7%). High and low molecular weight peptides had opposite change.	(Manus, Millette, Uscanga, et al. 2021)
L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2	Pea and rice	10% protein with 10^8 CFU/mL starter. Fermentation at 37 ± 1 °C for 14 ± 2 h.	In vivo digestibility, weanling male Wistar rats, aged of 20–23 days.	PER and NPR increased (from 1.88 to 2.32, 1.66 to 2.30). PDCAAS of PRF reached 100%, and true digestibility value approaching 90%, higher AD (87.64 ± 0.78) than PRNF.	(Manus, Millette, Dridi, et al. 2021)
L. crispatus BC4 and L. gasseri BC9	Soy	Inoculated at least 7 log CFU/mL. Fermentation at 42 ^oC, stored at 4 °C up to 28 days.	In vitro digestion according to the INFOGEST protocol	Fermented samples showed higher bioaccessibility. Protein hydrolysis rised from 423.8 to 592.1%.	(D’Alessandro et al., 2023)
P. acidilactici XZ31 and S. cerevisiae JM4	Wheat	150 g flour with 25 mL cell suspension (10^7 CFU/g dough). LAB and S in a ratio of 10:1. Fermentation at 30 °C for 24 h.	In vitro pepsin-trypsin digestion analysis, Caco-2 cell	Co-culture fermentation improved digestibility. FAA in L + S group increased, further decrease by about 60% and 98% in albumin/globulin and gluten.	(Fu et al., 2021)
L. plantarum HLJ29L2	Wheat	10^7 CFU/g flour. Primary fermentation at 35 °C for 19 h, and continued at 30 °C for 4 h	Simulated in vitro digestion methods	IVPD of DN-1 crackers was highest in the intestine and stomach (around 86%, 17%), higher content of Arg, Val, Phe, Leu.	(Hu et al. 2023)
W. confusa A16	Rapeseed	Fermentation at 25 °C for 24 h with ca. 6 log CFU/g strain.	IVPD according to the INFOGEST	RPC and RPI exhibited higher total FAA. EAA and NEAA were higher in RPC. RPI DSD (27.5%) enhanced IVPD than RPC DSD.	(Wang, Rosa-Sibakov, et al. 2022)
L. plantarum and S. cerevisiae	Maize	Fermentation at 37 °C for 12, 24, 36, 48 h with 1 × 10^6 cells/mL strains.	IVPD	Protein content and IVPD improved. Trypsin inhibitor content decreased (55.56→19.94 mg/100 g).	(Terefe, Omwamba, and Nduko 2021)
L. plantarum T6B10 and L. rossiae T0A16	Quinoa	Fermentation at 30 °C for 16 h with approx. log 7.0 CFU/g concentration.	IVPD	FQP increased IVPD (40.4 ± 0.1), attributed to proteolysis. CS (Lys, His, Leu, Cys, Thr, Met, Val) and NI were higher than WP.	(Lorusso et al. 2016)
L. plantarum DPPMAB24W	Faba bean	Fermentation at 30 °C for 24 h with ca. log 7.0 CFU/g concentration.	A multi-step protocol, in vitro gastrointestinal digestibility	IVPD, CS, EAAI, BV, PER, and NI of pasta containing 30% fermentation concentration improved.	(Carlo G. Rizzello et al. 2017)
L. plantarum T0A10	Black Chickpea	Dough cell density approx. 7.0 log10 CFU/g. Fermentation at 30 °C for 24 h.	In vitro digestion with pepsin followed by pancreatin	Fortified pasta IVPD up to 38%. Total FAA was the highest (602 mg/kg), other groups had lower concentrations (-72%, −29%).	(De Pasquale et al. 2021)
L. plantarum ST-III	Wheat and buckwheat	Fermentation at 30 °C for 24 h with 8.0 log CFU/g inoculation amount.	Pepsin-trypsin digestion procedure in vitro	EAA and NEAA increased by 22.22 and 16.92% in TBS. Degraded albumin in WS, TBS, and WTBS is 90%, 3% and 15.8%.	(Zhou et al. 2022)
L. plantarum MRS1 and L. brevis MRS4	Lentils, beans, chickpeas, and peas	Dough cell density ca. 7.0 log10 CFU/g. Fermentation at 30 °C for 24 h.	In vitro digestion in the GIT	Fermentation enhanced FAA contents in rF and gF up to 60%. IVPD of rF varied from 72 to 80% approximately (chickpeas and white beans).	(De Pasquale et al. 2019)

Note: L. acidophilus, Lactobacillus acidophilus; L. plantarum, Lactobacillus plantarum; L. casei, Lactobacillus casei; L. rhamnosus, Lactobacillus rhamnosus; L. lactis, Lactobacillus lactis; L. paracasei, Lactobacillus paracasei; L. bulgaricus, L. delbrueckii, Lactobacillus delbrueckii; L. crispatus, Lactobacillus crispatus; L. gasseri, Lactobacillus gasseri; L. mesalococcus, Leuconostoc mesenteroides; B. animalis, Bifidobacterium animalis; B. subtilis, Bacillus subtilis; B. coagulans, Bacillus coagulans; W. confusa, Weizmannia coagulans; S. thermophilus, Streptococcus thermophilus; S. cerevisiae, Saccharomyces cerevisiae; P. acidilactici, Pediococcus acidilactici.

AAN, α-amino nitrogen; TN, total nitrogen; AA, amino acid; AAS, amino acid score; FAA, free amino acid; FAN, free amino nitrogen; EAA, essential amino acid; NEAA, nonessential amino acid; SP, soybean protein/soluble protein; SPI, soybean protein isolate; FSPI, fermented soybean protein isolate; IPP, isolated pea protein; PRF, pea and rice proteins; L9SM, L. paracasei L9 soymilk; CSSM, commercial starter soymilk; GIT, gastrointestinal tract; FSB, fermented soybean meal; ATTD, apparent total tract digestibility; MFSM, mixture of fermented soybean meal and fish meal; RPC, rapeseed protein concentrate; RPI, rapeseed protein isolate; TBS, Tatary Buckwheat Sourdough; WS, Wheat Sourdough; WTBS, Wheat-Tartary Buckwheat Sourdough; AD, apparent digestibility; CS, chemical score; NI, nutritional index; EAAI, essential amino acid index; PER, protein efficiency ratio; NPR, net protein ratio; BV, biological value; PDCAAS, protein digestibility-corrected amino acid score.

2.3. Probiotics fermented plant proteins to promote their bioactivity

Plant proteins possess diverse applications as bioactive peptides (BAPs) due to their wide range of functional properties. These BAPs demonstrate various activities such as antitumor, antimicrobial, antihypertensive, and antioxidant effects. The generation and roles of BAPs involve different mechanisms (Kumar et al. 2021): (a) Protein hydrolysis leads to the formation of bioactive hydrolysates/BAPs. (b) The enzyme angiotensin-I-converting enzyme (ACE) plays a key part in transforming angiotensin-I into its active form. Antihypertensive BAPs regulate blood pressure through suppressing ACE activity. Angiotensin-II increases blood pressure by causing vasoconstriction. (c) BAPs are believed to indirectly enhance the formation of nitric oxide or the bioavailability of bradykinin, eventually leading to vasodilation. (d) BAPs play a role in controlling the levels of low-density lipoproteins (LDL) through interaction with LDL-receptors. (e) BAPs contribute to the sequestration of free radicals formed in mitochondria and other cellular organelles, exerting their antioxidant effects. (f) BAPs exhibit potent anticancer properties and can be used as effective agents against cancer cells. (g) Antimicrobial BAPs interplay with microbial membranes and cause the eventual cell death.

Because probiotics fermentation has a cutting effect on the peptide chain in plant proteins, the specific proteases and peptidases produced by probiotics fermentation can selectively cut specific sites in plant proteins. After the synergistic action of various enzymes, longer peptide chains in plant proteins are further broken down into shorter peptide segments, yielding a series of peptides with physiological activity (Rim et al. 2021). Such antioxidant peptides, blood pressure-lowering peptides, antibacterial peptides, and other active peptide products enhance their nutritional value and specific functionality so that they have better bioavailability and physiological activity and are easier to be absorbed and utilized by the human body. Bioactive proteins and peptides in plant species shown to enhance health features when ingestion, such as regulating the body’s immune system response, improving chronic inflammation, lowering cholesterol levels, controlling obesity, and affecting antioxidant activity. Some studies have found that soybean protein isolates and PEPs have an impact on the growth and metabolism. This suggests that through probiotics fermentation, soybean protein isolates can produce peptides that have a positive impact on probiotics (Zhang et al. 2020). By fermentation and other methods, soy protein can produce potential bioactive and functional peptides, such as arginine-rich PEPs and isoleucine-rich PEPs (Zhu et al. 2023).

Plant protein contains a variety of bioactive substances, which can provide effective prevention and control strategies for regulating the disorders of glucose and lipid metabolism, elevations in inflammation levels, disturbances in intestinal environment, and immune abnormalities through the biological pathway combined with probiotics (Schroeder et al. 2017). By combining Tartary buckwheat protein with Lactobacillus plantarum ST-III, it has been comprehensively analyzed the effect of the dietary intervention mode combined with the two on regulating the disorder of lipid metabolism in mice (Zhao 2019).

Studies have shown that proteolytic substances can improve digestion and absorption as prebiotics by improving intestinal permeability and intestinal immunity (Aloo and Oh 2022). It was explored the mitigating potential of plant protein-probiotic preparations on lead poisoning in mice. The group given plant protein-probiotic preparations showed lower lead content in blood, liver and kidney tissues, higher antioxidant enzyme activity (such as superoxide dismutase and glutathione peroxidase activity), and lower malondialdehyde content (Wang, Wu, et al. 2021). Additionally, the liver and kidney histopathological changes of these mice were also light, which laid the foundation for further research on the application of plant protein-probiotic preparations in the field of toxicology.

Generally, food fermentation positively affects probiotic proliferation and protein availability (Figure 2C). Nevertheless, it is crucial to acknowledge that fermentation can also reduce the presence of substances like phytic acid, protease inhibitors and tannin. This reduction could result in a variation in health-promoting activities (Kårlund et al. 2020). Table 3 illustrated different strains and plant proteins fermentation, and details experimental fermentation conditions, in vivo and in vitro experimental models and specific change indicators to prove the impact of fermentation of different plant proteins on nutritional functional properties and health-promoting activities.

Table 3. The combination of probiotics and plant proteins promotes their bioactivity.

Probiotics strain	Fermentation condition	Experimental model	Plant protein	Performance	Reference
W. confusa A16	Fermentation for 10/17/24 h at 20/25/30 ^oC, starter inoculum (5/6/7 log CFU/g).	Fermented sourdough, in vitro	Faba Bean	Higher temperature and longer fermentation elevated organic acids, esters and aromatic compounds.	(Tuccillo et al. 2022)
B. pseudocatenulatum INIA P815	Initial concentration 6.5–7.0 log CFU/mL Fermented at for 24 h at 37 ^oC.	C57BL/6J female mice model, in vivo	Soy	FSB provided benefits in lipid profile and fertility. Increased retrieved oocytes and zygotes, lower triglycerides (65.9 ± 21.0).	(Ruiz de la Bastida et al. 2023)
L. plantarum P1201	Inoculated at the level (2.0 × 10^7 CFU/mL. FSPHM sample fermented at 37 °C for 48 h.	Fermented soybean powder hydrolysate milk, in vitro	Soy	CLA, TPC and aglycone contents increased along with enhanced antioxidant properties (ABTS (87.6%), DPPH (77.4%), Hydroxyl (47.0%)).	(Lee et al. 2018)
L. brevis WCP02, L. plantarum P1201	Inoculated (2.0 × 10^7 CFU/mL) with 5% to produce SPY.	Fermented soybean yogurts, in vitro	Korea soybean	SPY exhibited enzymatic inhibition and antioxidant capacities. α-glucosidase, α-amylase, and pancreatic lipase increased, total average iso reduced (971.1 μg/g).	(Hwang et al. 2020)
L. plantarum	The cells mixed with 100 ml BPI, and final cell was 7 Log CFU/mL. Fermented at 37 °C for 15 h.	Fermented blood fruit, in vitro	Blood fruit (Haematocarpus validus Bakh.F. Ex Forman) seed	Fermented sample showed antimicrobial, anticancer, antidiabetic properties. HSP82 is the unique protein belongs to HSP90 family.	(Sasikumar, Vivek, and Jaiswal 2021)
L. plantarum TK9 and L. paracasei TK1501	TBW/water was 1:1.5, 10^7 CFU/g. Fermented at 37 °C for 24 h.	Solid-State Fermented (SSF) TBW, in vitro	Tartary Buckwheat (TBW)	SSF TBW exhibited antioxidant and antidiabetic properties. The highest α-glucosidase and DPP-IV inhibition (IC50, 0.51 mg/mL TK9, 2.47 mg/mL TK1501).	(Feng et al. 2018)
L. casei subsp. casei R-68 and L. plantarum 1 R 1.3.2	10% honey, 5% R-68 and 5% 1 R. 1.3.2 in soymilk. Incubated at 37 °C for 18 h.	Postmenopausal women, in vivo	Soy	SMH Lp group reduced osteocalcin levels (27.74) at beginning and lead to SCFA and calcium absorption enhanced.	(Desfita et al. 2021)
Water kefir grains (WKG)	1 % w/v QPCs with 5 % (v/v) WKG for 5 days at 25 ^oC.	Fermented quinoa protein concentrates (QPCs), in vitro	Quinoa seeds	TPC increased to 528.8 mg of GAE/100 g. All phenolic compounds measured increased, epicatechin being the most abundant (97.48 ± 1.41, Day2).	(Alrosan et al. 2022)
Kefir	Soymilk inoculated with 5% (w/v) kefir for 24 h fermentation (FSM).	Wistar female rats, in vivo	Soybeans	FSM could protect from hepatic and kidney tissues toxicities. Intestinal and pancreas lipase activity, TC, LDL-c and blood glucose decreased by 34, 35, 24, 66 and 36%.	(Tiss et al. 2020)
B. siamensis, B. tequilensis, B. velezensis, and L. lactis subsp. lactis	Soybean meal (SM) with 1 × 10^7 CFU/g of BS, BT, BV, and LL.	Bullfrogs (Lithobates catesbeianus), in vivo	Soybean	Fermentation improved immune response and intestinal barrier. In BT and LL groups, showed higher C4, IgM, IL-10, ZO-1 level, abundances of Bacillus, Cetobacterium.	(Wang, Wu, et al. 2021)
L. plantarum DSM 20174	9% (w/w) PPI dispersion in DI water with 7 Log CFU/mL L. plantarum.	Rabbits, in vivo	Pea (Pisum sativum L.)	Treated samples reduced immunogenicity and tolerated better by allergic or sensitized individuals. The foaming capacity increased (1190–2575%) and reduced off-flavors.	(García Arteaga et al. 2022)
L. paracasei MIUG BL2	5% pork lard or 2% (w/v) freeze-dried okara.	SSF Pork Lard and Okara, in vitro	Okara	SSF highlighted antioxidant and antimicrobial properties. DPPH was 6.77-17.78 mM TE/g, against A.niger ranged from 24 to 64%.	(Cotârleț, Stănciuc, and Bahrim 2020)
B. subtilis DSM 10, L. helveticus DSM20075, S. cerevisiae TMW 3.210	1 × 10^8 CFU/mL of bacteria. Fermentation for 24 and 48 h, stopped by heat treatment at 90 °C for 20 min.	Fermented SPI, in vitro	Soybeans	L. helveticus revealed the most abundant reduction in immunoreactivity. Foaming activity was nearly doubled, decreased emulsifying capacity and off-flavors.	(Meinlschmidt et al. 2016)
Lactobacillus (ACCC10637), B. licheniformis 1.0813	Inoculation amount, 5% (v/w), Lactobacillus 8.5 × 10^6 CFU/g; B. licheniformis 6.8 × 10^6 CFU/g. Fermented rapeseed meal (FRSM) for 72 h at 30 ^oC.	Male yellow-feathered broiler chickens, in vivo	Rapeseed	FRSM increased the productivity and bioavailability. ADG and DFI values were higher, ALT and BUN levels were lower than RSM (p < 0.05).	(Wang et al. 2019)
B. subtilis YY-1 and S. cerevisiae YY-2	5 × 10^6 CFU/g S. cerevisiae and 2 × 10^6 CFU/g B. subtilis. Fermented extruded-pretreatment rapeseed meal (FERSM) for 72.03 h at 32.5 ^oC.	Wistar rats (half male and female, weaned stage), in vitro and in vivo	Rapeseed	FERSM improved antioxidant properties. Higher T-SOD, GSH and DPPH radical scavenging activity and lower MDA levels (p < 0.05).	(Wang, Rosa-Sibakov, et al. 2022)
L. sakei ssp. carnosus DSM 15831, L. amylolyticus TL 5 and L. helveticus DSM 20075	5% hydrolyzed (w/w) Lupin protein isolate (LPI) with 10^7 CFU/mL. Fermented at 37 °C and 42 °C for 24 h without stirring.	Fermented LPI, in vitro	Lupin (Lupinus angustifolius L. cultivar Boregine) seeds	Fermented samples degradated IgE-reactive polypeptide. A reduction in the major allergen Lup an 1 below 0.5% than unfermented (100%).	(Schlegel et al. 2021)
L. plantarum CCFM8661	Grape seed extract 3.6%, Freeze-dried bacterial powder 40%. At 4 g/20 g body weight via gavage.	Six-week-old male C57black/6 mice, in vivo	Grape seed	Dietary supplements can against Pb toxicity. Decreased Pb and recovered ALAD, SOD, CAT GSH, ZPP, MDA, the learning and memory ability.	(Zhai et al. 2018)
L. plantarum DSM-20174, L. perolens DSM-12744, L. fermentum DSM-20391, L. casei DSM-20011, Lc. cremoris DSM-20200, P. pentosaceus DSM-20336	A 9% (w/v) PPI dispersion. Fermented pea protein isolate (PPI) for 24 h and 48 h at 30 °C under anaerobe or aerobe.	Fermented PPI, in vitro	Pea	The protein solubility and emulsifying capacity decreased might cause a reduction of the allergenic protein fractions in PPI.	(García Arteaga et al. 2021)
L. plantarum 70810, Y-1, B1-6, ZR, M-6, MB1-6, L3-4 and Dong	1 mg/mL SPI with 10^8 CFU/mL strains. Fermented SPI for 48 h at 37 ^oC.	Fermented SPI, in vitro	Soybean	L. plantarum proved efficacy in lowering IgE reactivity (by 83.8%-94.8%) and SP content (53.8%-92.8%).	(Rui et al. 2018)
L. plantarum subsp. plantarum P15 and E. faecalis ZZUPF95	LAB inoculation was added at 2%. Fermented soybean meal (FSBM) for 30 days.	FSBM, in vitro	Soybean	P15 + ZZUPF95 had lower harmful bacteria and pH value (4.51), higher lactic acid level (21.25 mg/g), the most significant degradation in CF.	(Ma et al. 2022)
L. plantarum B1-6	3% (v/v) L. plantarum B1-6. Fermented at 37 ^oC, terminated at pH 7.0, 6.0, 4.5, 4.0, 3.5.	Fermented Soy Glycinin, in vitro	Soy	The largest particles and lowest immunoreactivity were produced by 1% glycinin. IgE reactivity reached 0.1-0.24% at FT-pH 4.5.	(Liu, Wang, and Wu 2021)
L. plantarum DPPMAB24W	Cell density ca. 7.0 log CFU/mL with flour in a ratio of 50:50 (wt:wt). Fermented at 30 °C for 48 h.	Fermented faba bean doughs, in vitro	Faba bean (Vicia faba L. major)	Antioxidant activity increased ca. 11%. TPC ranged from 1.10 ± 0.07 to 1.84 ± 0.18 mmol/kg. Antinutritional factors concentrations decreased.	(Verni et al. 2019)
L. plantarum KCCM 11322 and Bacillus species SJ-19	1 × 10^8 CFU/g ProA and ProB, Soybean meal 12.8%, Corn gluten meal 5.0%, Wheat gluten 5.85%, Soy protein concentrate 5.85%.	Juvenile olive flounders, in vivo	Soybean, corn, wheat	Dietary supplements enhanced intestinal microbial diversity, digestive enzyme activity, intestinal immune (IL-1β, IL-6, TNF-α, IL-10), resistance to pathogens (Streptococcus iniae).	(Jang et al. 2019)
S. thermophilus and L. delbrueckii subsp. bulgaricus	Inoculated with 3% starter. Fermented at 42 ± 1 °C to pH 4.6.	Fermented milks beverage, in vitro	Soy, pea, wheat and rice	Plant protein additives improved the nutraceutical properties and nutritional value. The viscosity level showed an increase (p < 0.01).	(Akin and Ozcan 2017)
Not mentioned	250g foxtail millet flour (FMF), 10^4-10^5 CFU/mL bacterial cell. Fermented at 38 °C for 20 h.	G-F-FMF, in vitro	Foxtail millet (Setaria italica L.)	G-F-FMF reduced anti-nutritional factors (phytate, tannin), enhanced bioactive constituents and antioxidant.	(Sharma and Sharma 2021)
L. plantarum C48 and L. brevis AM7	Initial cell density in sourdoughs of 7.0 log CFU/g for each strain. Incubated at 30 °C for 24 h.	Fermented sourdough, in vitro	Kidney bean, chickpea, grass pea, lentil, pea	TP, phytase and antioxidant activities increased; raffinose and condensed tannins concentrations decreased. GABA increased up to 624 mg/kg.	(Curiel et al. 2014)
L. plantarum DPPMAB24W	Fermented dough (FF) with 7.30 log CFU/g initial cell density. Incubated at 30 °C for 48h.	ex-vivo assays on human blood	Faba bean	Decomposition of vicine and convicine was more efficient following 48 h treatment in FF, and the hemolysis was 54 and 40% lower than Ct.	(Carlo Giuseppe Rizzello et al. 2016)
P. pentosaceus I02	30% faba bean flour with ca. log 6.0 CFU/g. Fermented at 20 °C for 24 h.	Faba bean Sourdough Bread (FSB), in vitro	Faba bean	Decrease the value of HI and predicted GI (81%, 84.2%), better protein biological value and nutritional indexes in FSB.	(Coda et al. 2017)
L. rossiae LB5, L. plantarum 1A7 and L. sanfranciscensis DE9	Fermented at 30 °C for 24 h with ca. 7.0 Log CFU/g LAB, mixed starter (ratio 1:1:1).	Sourdough fermented sprouted flours (sS), in vitro	Wheat, barley, chickpea, lentil and quinoa grains.	sS had higher GABA, total phenols and antioxidant activity than rS. Fermented quinoa led to the highest GI (76.4 ± 0.5).	(Montemurro et al. 2018)
L. plantarum DSM 32248 and L. rossiae DSM 32249	Final cell density was ca. 10^7 CFU/g. Fermented milling by-products bread (FMPB)contain 15% (w/w) of FMP. Fermented at 30 °C for 24 h.	Healthy nonsmoking volunteers, in vivo	Wheat germ and bran	High content of dietary fiber, nutritional indexes and low HI (55%) and GI (36.9%), which reduces the risk of T2 DM.	(Pontonio et al. 2017)
L. pentosus 9D3	Final cell density approx. 7 log CFU/mL. Fermented at 30 °C for 16 h.	Fermented brown rice milk (BRM), in vitro	Brown rice RD41	With high GABA (14.3 mg/100 mL), TPC (15.23 mg GAE/100 mL) and antioxidant activities, inhibit obesity and diabetes-associated enzymes.	(Suwapat et al. 2021)
L. plantarum 299v	Fermented at 37 °C for 20 h with 20 × 10^9 CFU/mL L.plantarum 299v.	Lupine and chickpea germinated fermented (LGF, CGF), in vitro	Lupine and chickpea	Fermentation caused bioactive compounds enhanced. It showed higher TPC, TFC (p < 0.05), and antioxidant activity.	(Adriana Dalila et al. 2023)
L. paracasei A1 2.6 and P. pentosaceus GS·B	Initial cell count of approx. 10^6 CFU/g. Fermented overnight at 30 ^oC.	Fermented quinoa/buckwheat soup, in vitro	Quinoa and buckwheat grains	Fermented quinoa cooked seeds were more efficient in increasing antioxidant capacity and TPC (74.8 ± 3.8).	(Rocchetti et al. 2018)
Lactococcus, Pediococcus, and Weissella	Raw cashews with the starter rejuvelac culture. Fermented for 2–4 days.	Fermented Cashew Cheese-Like Product, in vitro	Quinoa, cashew (Anacardium occidentale).	Fermentation significantly decreased cashew allergenicity.	(Chen et al. 2020)
FD-DVS YC-180 Yo-Flex^® (L. delbrueckii subsp. Bulgaricus, L. delbrueckii subsp. lactis, S. thermophilus)	Fermented germinated brown rice for 96 h (F-GBR96) with 2 × 10^7 CFU/g. Fermented at 42 °C until pH 4.4 ± 0.2.	F-GBR yogurt-like product, in vitro	Brown rice (BR)	Fermentation evaluated biological activity. F-GBR96 exhibited higher TPC, GABA, ORAC, ACE-inhibitory activity.	(Cáceres et al. 2018)
B. coagulans VHProbi C08	40 g soy protein concentrate, inoculated with 1.0% (v/v) starter culture. Fermented at 40 °C for 16 h.	Fermented plant- based drink, in vitro	Soy	Fermentation exhibited higher antibiotic and antioxidant activity, and the main metabolites were significantly different.	(Peng et al. 2021)
B. longum subsp. infantis CECT 4551 and L. plantarum CECT 220	450 g red quinoa with 1% (v/v) LP (QLPBB) or BL (QBLBB). Fermented at 37 °C for 24 h.	Probiotic red quinoa drinks, in vitro	Red quinoa (Chenopodium quinoa, L.)	The antioxidant activity and TPC were significantly increased in vitro digestion, which would improve bioactive compounds.	(Cerdá-Bernad et al. 2021)
L. fermentum CQPC04	The final viability count in the fermented soy milk was 1.3 × 10^8 CFU/mL. Fermented at 37 °C for 12 h.	Female ICR mice, in vivo	Soy	It could delay oxidative aging. Increased free soy isoflavones, peptides, GSH, GSH-Px, T-SOD, CAT, Col I, HA, and Col III, decreased NO, MDA, AGEs and H2O2.	(Zhou et al. 2021)
L. plantarum 299v	5.7 g henolea^® with L. plantarum (∼2 × 10^9 CFU/mL) until form coherent mass.	L. plantarum combine olive leave extract (Lac-Phe), in vitro	Olea europaea leaves (Phenolea^®)	Lac-Phe granules confirmed probiotic alive into colon, against adverse conditions. Boosted oleuropein bioconversion to hydroxytyrosol.	(Aponte et al. 2018)
L. p lantarum CRL2211, W. paramesenteroides CRL2182	DY160, L. plantarum and/or W. paramesenteroides 7 log CFU/g. Fermented at 37 °C for 24 h.	Fermented chickpea, in vitro	Chickpea	TPC, tannin and trypsin inhibitor removal (525.00 mg GAE/100 g, 28.80 mg GAE/100 g and 1.60 mg/g) higher in LAB dough.	(Sáez et al. 2021)
L. paracasei CD4	The soybeans with water (1:10), inoculated at 1% level. Fermented at 37 °C for 24 h.	Fermented soymilks, in vitro	Soy	A decrease in pH and TPC was observed (p < 0.05), the antioxidant activity TEAC and TA was enhanced during 21 days storage.	(Choudhary et al. 2018)

Note: L. plantarum, Lactobacillus plantarum; L. brevis, Lactobacillus brevis; L. casei, Lactobacillus casei; L. paracasei, Lactobacillus paracasei; L. helveticus, Lactobacillus helveticus; L. fermentum, Lactobacillus fermentum; L. sakei, Lactobacillus sakei; L. amylolyticus, Lactobacillus amylolyticus; L. perolens, Lactobacillus perolens; L. pentosus, Lactobacillus pentosus; L. rossiae, Lactobacillus rossiae; B. pseudocatenulatum, Bifidobacterium pseudocatenulatum; B. licheniformis, Bacillus licheniformis; B. subtilis, Bacillus subtilis; B. siamensis, Bacillus siamensis; B. tequilensis, Bacillus tequilensis; B. velezensis, Bacillus velezensis; A.niger, Aspergillus Niger; W. confusa, Weizmannia coagulans; W. paramesenteroides, Weissella paramesenteroides; S. cerevisiae, Saccharomyces cerevisiae; S. thermophilus, Streptococcus thermophilus; E. faecalis, Enterococcus faecalis.

DPP-IV, dipeptidyl peptidase IV; CLA, conjugated linoleic acid; TPC, total phenolic content; ABTS, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid); DPPH, 2,2-diphenyl-1-pycrylhydrazyl; ALAD, δ-aminolevulinic acid dehydratase; T-SOD, total superoxide dismutase; CAT, catalase; GSH, glutathione; MDA, malondialdehyde; TEAC, trolox equivalent antioxidant capacity; NO, nitric oxide; HA, hyaluronic acid; H₂O₂, hydrogen peroxide; Col, collagen; AGE, advanced glycation End products; ZPP, zinc protoporphyrin; TFC, total flavonoid content; SCFA, short chain fatty acid; TC, total-cholesterol; LDL-c, low-density lipoprotein cholesterol; C4, complement 4; Ig, immunoglobulin; IL, interleukin; TNF-α, tumor necrosis factor-α; ZO-1, zonula occludens-1; ADG, average daily weight gain; DFI, daily feed intake; ALT, alanine aminotransferase; BUN, blood urea nitrogen; GABA, γ-aminobutyric acid; T2 DM, type 2 diabetes mellitus; ORAC, oxygen radical absorbance capacity; ACE, angiotensin I-converting enzyme.

3. Mechanisms of plant proteins-probiotics interactions

It has been widely accepted that probiotics fermentation products can alleviate or prevent various diseases by regulating intestinal health, establishing a close connection between probiotics and host health (Mazziotta et al. 2023). Probiotics, known for their ability to balance intestinal microecology, promote digestion and absorption, enhance gastrointestinal motility, boost body immunity, and alleviate allergic reactions, have been recognized as guardians of health (Sumera, Aqsa, and Imtiaz 2023). Research suggests that probiotics exert their beneficial effects through mechanisms such as immune response modulation, prevention of physiological stress, inhibition of pathogens, microbiota balance, and improvement of intestinal barrier function (Suez et al. 2019). Well-known probiotic microorganisms with a long history of use include Lactococcus, Lactobacillus, Streptococcus, Enterococcus, and Bifidobacterium.

Probiotics play a crucial role in regulating the gut microbiota, which in turn affects gut bacteria involved in proteolysis (Salam et al. 2023). By breaking down large molecular plant proteins into simpler forms, probiotics enhance the digestion and absorption of these nutrients in the human body. This metabolic process also leads to the production of various substances including short-chain fatty acids (SCFAs), exopolysaccharides, and vitamins. When it comes to the degradation of hard-to-digest plant proteins found in food, probiotics have the ability to break them down into smaller peptides and amino acids (Figure 3), thereby improving their digestibility and nutritional value (Wang, Wu, et al. 2021).

Figure 3. Health promotion mechanisms of plant proteins-probiotics interactions.

Th1 cell, type 1 T helper cell; Th2 cell, type 2 T helper cell; l; Th17 cell, type 17 T helper cell Treg cell, regulatory T cell; sIgA, secreted immunoglobulin A; PPR, pattern recognition receptor; MUC, mucins; IL, Interleukin; TGF-β, transforming growth factor beta; TLR, toll-like receptor; LPS, lipopolysaccharide; ROS, reactive oxygen species.

The combination of plant proteins with probiotics can effectively optimize the dietary nutrient structure needed by the human body, catering to the diverse requirement and offering health advantages (Adriana Dalila et al. 2023). Furthermore, the post-epidemic era has witnessed a rise in the prevalence of gastroenteritis and enteropulmonary diseases, with various health symptoms indicating the need for the consumption of high-quality protein to maintain muscle mass, boost immunity, and enhance physical fitness.

The interactions mechanisms between probiotics and plant proteins are roughly manifested in the following aspects:

1. Changes in plant protein solubility: The binding of probiotics to plant proteins may result in changes in plant protein solubility.

2. Promote plant proteins degradation: Probiotics have protease activity, which can promote the degradation of protein macromolecular aggregates into small peptides or polypeptides, thereby increasing the content of α-helix structure. The increase in this structure may lead to modifications in the function, improve the solubility and stability of proteins, and make it easier to digest and absorb.

3. Formation of complex: The combination of probiotics and plant proteins may form a complex, which may have new bioactivities such as antioxidant, anti-inflammatory, antibacterial, anti-allergen and immune regulation, and can also affect the combination of pathogenic bacteria and IECs, degrade anti-nutritional factors, and improve the bioavailability of proteins.

4. Change plant protein function: The combination of probiotics with plant proteins may change protein function. Some studies have shown that probiotics can improve the fermentation performance of plant proteins, change the emulsification, foaming, gel formation ability of proteins, etc., and improve their application value in food processing.

Plant proteins and probiotics engage in interactions such as adhesion, binding, and co-metabolism. As a signal molecule for cell attachment, proteins can bind to receptors on the surface of probiotics. This binding activates signal pathways within the cell, which subsequently affects cell attachment, growth metabolism, and probiotic function (do Carmo et al. 2018). Adhesion and binding represent significant mechanisms by which probiotics attach to the intestinal wall in the host intestine. Surface proteins enable probiotics to bind to IECs, facilitating their attachment to intestinal cells and enabling them to perform their functions. These interactions between proteins and probiotics, as well as co-metabolism, play a fundamental role in maintaining a balanced gut microbial community (Kieliszek et al. 2021).

Plant proteins combined with probiotics can produce many synergistic effects (Figure 3), including the following aspects:

1. Increase the nutritional value: The combination of probiotics and plant proteins can improve the bioavailability of protein, promote the digestibility of protein and the growth of probiotics, and increase its nutritional value to the body.

2. Regulate the body’s immunity: proteins combined with probiotics can participate in the process of regulating the function of the immune system and promote the body’s resistance to diseases.

3. Maintain the intestinal health: Probiotics can maintain the balance of the intestinal microbiota, and protein combined with probiotics may be more easily absorbed and utilized by the intestine, contributing to intestinal environmental homeostasis.

4. Improve the plant protein stability: the combination of plant proteins and probiotics can improve the stability of plant proteins and extend their shelf life, thereby increasing the availability of related products.

Plant proteins in the diet encompass more than just protein; they also consist of polyphenols, minerals, fiber, and other components, creating a complex matrix. Fermented plant protein food ingredients contain various constituents that are transported to the gut, exerting an impact on both intestinal and overall health (Figure 3). Fermentation enhances the bioavailability and absorption of polyphenols, minerals, and amino acids (Arghya et al. 2023; Vincenzo et al. 2024). Fermented plant protein foods offer a range of amino acids and derivatives that can function as neurotransmitters and/or immune transmitters. Additionally, these foods contain beneficial microorganisms and bioactive peptides that can inhibit the growth and colonization of intestinal pathogens. The immune system is influenced by organic acids, polysaccharides, and SCFAs, which are either directly delivered to or produced by gut microbes. These substances contribute to maintaining the integrity of the gut barrier, suppressing pathogens, and reducing inflammation (Wastyk et al. 2021). To gain a deeper understanding of the underlying mechanisms, randomized controlled trials and large cohort studies are necessary.

4. Challenges and perspectives

Probiotics fermentation can modify the physicochemical properties and health functions of food, resulting in distinct flavors and diversified taste and texture of plant protein-based foods. By considering the synergistic interaction between plant proteins and probiotic strains, various beneficial outcomes can be achieved, including the enrichment of active functional substances, extraction of effective components, release of flavor compounds and metabolites, generation of novel active ingredients, reduction of unpleasant flavors, and improved biological utilization (Figure 4). Applying probiotics fermentation to various plant proteins can enhance the overall value of food products.

Figure 4. The research framework of the challenges and perspectives for plant proteins-probiotics interactions.

Moreover, the combination and fermentation of different probiotic strains lead to sensory and biological characteristics that are distinct to each product. It is worth to note the importance of making full use of sensory analysis research in fermented products. By designing an effective analysis program, ensure that the composition, odor and texture of the final fermented product do not produce negative effects (Gámbaro and Matthew 2020). This approach contributes to the specific biological and chemical composition of fermented foods and enhances the diversity of fermented products, thereby increasing their acceptance among consumers.

Currently, the regulatory impact of probiotics on gut microorganisms and their ability to maintain intestinal homeostasis remain uncertain. The stability and survival rate will be challenged in the process of processing and digestion and the resilience of probiotic preparations to harsh conditions in the gastrointestinal tract requires enhancement. Typically, the mucosa’s adhesion in the colon is insufficient, leading to poor bioaccessibility of probiotic preparations and hindering their effectiveness. The selective adhesion and colonization of probiotics in specific tissues like inflamed colon are yet to be investigated.

In the future, the health benefits of combining plant proteins with probiotics will be further strengthened, to achieve the advancements in plant protein raw materials, probiotics, and their production processes. The selection of probiotic strains is a crucial prerequisite for fermenting plant proteins. Given the wide range and variety of plant proteins and probiotic sources, it is necessary to screen specific probiotic strains suitable for fermentation based on the different properties of plant proteins. In the above process, strain screening, functional verification and high-activity preparation technology, even including the industrialization process of the final fermentation product, may face a lot of technical investment and research costs. Additionally, it is important to explore the fermentation process of different strains, design precise fermentation control technologies, and simplify the fermentation process to efficiently prepare substances in the fermentation system.

The development of functional foods, particularly those incorporating plant proteins and probiotics, remains an intricate and ongoing endeavor. Ongoing research is essential to accumulate scientific evidence that links these components to host nutrition and health. A comprehensive investigation of the mechanisms by which probiotics interact with plant proteins calls for further exploration. As shown in Figure 4, the research framework is highlighted to clarify challenges and perspectives for plant proteins-probiotics interactions. It is crucial to characterize the structure of selected plant proteins, perform high-throughput sequencing screening of probiotic strains, and conduct safety studies. Furthermore, it is important to study the effects of probiotics on the digestion and absorption of plant proteins, gain a better understanding of the benefits and potential mechanisms of fermented plant proteins in promoting gut health, and explore the interactions mechanisms between plant proteins and probiotics.

In addition, due to individual variations and the intricate nature of probiotics and plant proteins, additional research is required to devise clinically efficacious combinations. Fermented foods offer a valuable amalgamation of beneficial microorganisms and bioactive compounds. By scientifically combining probiotics and plant proteins, appropriate concentrations and dosages can be selected to achieve synergistic effects, thus optimizing their incorporation and expanding their applications in the realm of health. However, it must be emphasized that the success of the laboratory scale does not mean that it can scale up production, so there are huge economic challenges to achieve the expansion from the laboratory to industrial production, such as technology maturity, large-scale production costs, market demand, results transformation, talent support, etc. Furthermore, to ensure consistent quality in plant proteins-based fermented foods, comprehensive technical standards and quality criteria need to be developed for evaluating the final food products.

Furthermore, probiotic fermentation and metabolism processes are intricate. To ensure the targeted delivery of probiotics, the use of precise control of the release mechanism and the selection of appropriate packaging technology have become critical. The research on the processing technology of composite products combining plant proteins and probiotics is still in its early stages. The interaction mechanisms among different combinations of plant proteins and probiotics are not fully understood, and the coexistence of these components in the fermentation system presents challenges for product separation and purification. Additionally, compelling evidence is essential to validate the benefits and mechanisms of probiotics and plant proteins on nutrition and health, emphasizing the importance of randomized and controlled human trials. The potential of plant protein-probiotic interactions remains largely unclear, necessitating continuous efforts to explore the diversity and possibilities of fermented plant proteins products, and achieve innovative advancements in their combination.

Author contributions

Conceptualization, Jun Du, Caili Fu, and Dejian Huang; writing—original draft preparation, Juntao Kan, Qiming Wu and Zhengying Cui; writing—review and editing, Jun Du, Caili Fu, Dejian Huang and Lin Chen; resources and visualization, Yuchen Ma, Xin Liu, Ruifang Dong and Lin Chen. All authors have read and approved the manuscript.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by [Science and Technology Project of Jiangsu Province #1] under Grant [number BZ2022056]; [the Key Research and Development Project of Hainan Province #2] under Grant [number ZDYF2022XDNY335]; [the Cooperation project of Amway China Co., Limited and the National University of Singapore (Suzhou) Research Institute #3, #4] under Grant [No. Am20220229RD, Am20230595RD].