Application of the Reuterin System as Food Preservative or Health-Promoting Agent: A Critical Review

The reuterin system is a complex multi-component antimicrobial system produced by Limosilactobacillus reuteri by metabolizing glycerol. The system mainly includes 3-hydroxypropionaldehyde (3-HPA, reuterin), 3-HPA dimer, 3-HPA hydrate, acrolein and 3-hydroxypropionic acid, and has great potential to be applied in the food and medical industries due to its functional versatility. It has been reported that the reuterin system possesses regulation of intestinal flora and anti-infection, anti-inflammatory and anti-cancer activities. Typically, the reuterin system exerts strong broad-spectrum antimicrobial properties. However, the antimicrobial mechanism of the reuterin system remains unclear, and its toxicity is still controversial. This paper presents an updated review on the biosynthesis, composition, biological production, antimicrobial mechanisms, stability, toxicity and potential applications of the reuterin system. Challenges and opportunities of the use of the reuterin system as a food preservative or health-promoting agent are also discussed. The present work will allow researchers to accelerate their studies toward solving critical challenges obstructing industrial applications of the reuterin system.


Introduction
In recent years, negative public perception of chemical preservatives prompts an increasing preference of consumers for "natural" agents from biological systems as alternatives [1,2]. To meet the consumers' demand for "natural preservatives", essential oils extracted from plants [3,4], bacteriocins from lactic acid bacteria such as nisin or pediocin PAß1-AcH and bacteriocin-producing protective cultures such as Carnobacterium maltaromaticum UAL307 (Micocin ® [5,6]) are used commercially as food preservatives. However, high production cost, narrow antimicrobial spectrum and limited antimicrobial activity challenge the application of these "natural preservatives" [2]. Further improvement in food safety and quality still necessitates the development of novel antimicrobial agents from natural origins.
The reuterin system is a multi-component antimicrobial complex produced by Limosilactobacillus reuteri by metabolizing glycerol. Reuterin was first discovered in the 1980s by Axelsso et al. [7], who observed that resting cells of L. reuteri converted glycerol into a low-molecular-weight, non-protein, antimicrobial substance, which was later referred as reuterin. Further research verified that reuterin is a dynamic multi-compound equilibrated system consisting mainly of 3-hydroxypropionaldehyde(3-HPA), its dimer, its hydrate and acrolein in aqueous solution [8,9]. Reuterin generally refers to 3-HPA, because 3-HPA is not only the precursor of other reuterin compounds but also considered to be the main antimicrobial component in the system. Among the reuterin-producing bacteria, including Bacillus, Klebsiella, Citrobacter, Enterobacter, Clostridium and Lactobacillus [10], the food-grade L. reuteri strains are the most suitable for reuterin production at industrial scale due to

Biosynthesis and Composition of the Reuterin System
L. reuteri converts glycerol, as the electron acceptor, into an intermediate compound 3-HPA by the coenzyme B 12 -dependent glycerol dehydratase (GDH) in cells under anaerobic conditions. GDH exists as a dimer of subunits α2, β2 and γ2. The nucleotide and amino acid sequences of GDH are highly conserved in L. reuteri [24,25]. After being excreted outside the cell, 3-HPA forms a multi-compound dynamic reuterin system in aqueous solution, which includes 3-HPA, 3-HPA hydrate, 3-HPA dimer and acrolein. Moreover, 3-HPA in the cell is further reduced to 1,3-propanediol (1,3-PD) by the 1,3-PD oxidoreductase (an NADH-linked dehydrogenase) and spontaneously transformed into a structural isomer of lactic acid, 3-hydroxypropionic acid , in three stages. First, 3-HPA is converted to 3-hydroxypropionyl-CoA (3-HP CoA) by the propionaldehyde dehydrogenase. Second, 3-HP CoA is phosphorylated to 3-hydroxypropionyl phosphate (3-HP-P) by the phosphate propanoyltransferase. Finally, dephosphorylation of 3-HP-P leads to the formation of 3HP catalyzed by the propionate kinase [26]. Notably, 1,3-PD and 3-HP can also be secreted [12]. A detailed metabolic pathway of reuterin and the components of the reuterin system are shown in Figure 1.
Gene islands are clusters of genes that are likely to be associated with the horizontal gene transfer events in bacteria. The genomic island in L. reuteri is believed to be related to the evolution of its probiotic properties in the human gut. The complicated biosynthesis of reuterin is determined by a genomic island containing a 1,2-propanediol utilization (pdu) cluster together with a vitamin B 12 cluster (namely the pdu-cbi-cob-hem gene cluster) [27], which is proposed to be transcriptionally arranged as seen in Figure 2. The island harbors the pocR gene (lreu_1750)-encoding protein PocR that participates in both glycerol utilization and B 12 biosynthesis [28]. The ability of L. reuteri to produce vitamin B 12 is generally considered to be associated with 3-HPA production. Biosynthesis of 3-HPA from glycerol by vitamin B 12 -dependent glycerol dehydratase is encoded by gupCDE (pduCDE) in the pdu operon [27]. The pduF gene encodes a hydrophobic protein that facilitates the diffusion of glycerol [29]. The pduL, pduP and pduW confer the transformation of 3-HPA to 3-HP [30]. The gene sets (cbi, cob and hem) that are involved in cobalamin biosynthesis are adjacent to the pdu operon [27]. in the pdu operon. [27]. The pduF gene encodes a hydrophobic protein that facilitates the diffusion of glycerol [29]. The pduL, pduP and pduW confer the transformation of 3-HPA to 3-HP [30]. The gene sets (cbi, cob and hem) that are involved in cobalamin biosynthesis are adjacent to the pdu operon [27].   [27,28]. Yellow, pdu including gupCDE genes; Purple, cob genes; Green, cbi genes; Blue, hem genes; Red, pocR. The pocR gene regulates the synthesis of reuterin and cobalamin. in the pdu operon. [27]. The pduF gene encodes a hydrophobic protein that facilitates the diffusion of glycerol [29]. The pduL, pduP and pduW confer the transformation of 3-HPA to 3-HP [30]. The gene sets (cbi, cob and hem) that are involved in cobalamin biosynthesis are adjacent to the pdu operon [27].   [27,28]. Yellow, pdu including gupCDE genes; Purple, cob genes; Green, cbi genes; Blue, hem genes; Red, pocR. The pocR gene regulates the synthesis of reuterin and cobalamin.  [27,28]. Yellow, pdu including gupCDE genes; Purple, cob genes; Green, cbi genes; Blue, hem genes; Red, pocR. The pocR gene regulates the synthesis of reuterin and cobalamin.

Biotechnological Production of Reuterin
Reuterin (3-HPA) is the precursor of the reuterin system's components. Although 3-HPA can be produced by chemical synthesis with acrolein dehydration, the reaction has to be completed at high temperature, high pressure and relatively high cost [10]. Biological production of 3-HPA has the advantage of mild reaction conditions, high yield and low cost compared with chemical synthesis [10]. Production of 3-HPA by fermentation is carried out by the in situ process or the two-step process. The former is usually achieved by metabolizing glycerol by L. reuteri in MRS medium, but low yield and a complicated purification process limit its application. The latter involves preparing a crude 3-HPA solution by a simple two-step process followed by purification [11]. Specifically, a large number of whole cells of L. reuteri are firstly collected from the MRS medium. Secondly, washed cells are incubated in a glycerol-water solution to produce 3-HPA. Finally, 3-HPA is purified by ion exchange resin, chromatography or reactive distillation methods [32][33][34][35].
There are some factors that affect the production of 3-HPA in the two-step process, such as glycerol concentration, biomass, incubation time, incubation temperature, etc. Bauer et al. [36] studied the effect of glycerol concentration (100~400 mM) on 3-HPA production and found that 300mM glycerol had the highest 3-HPA yield (27 mM) at a resting cell concentration of 1 × 10 9 CFU/mL after 120 min of incubation at 20 • C, indicating that high concentrations of glycerol possibly inhibited the glycerol dehydratase activity of resting cells. L. reuteri was immobilized in spherical calcium alginate beads to increase 3-HPA productivity [37]. Moreover, the production of 3-HPA was expedited at pH 6, while no significant difference was found at incubation temperatures of 15 • C and 37 • C. Addition of lactate in the glycerol-water solution had a positive impact on the accumulation of 3-HPA while glucose showed the opposite result [38]. However, Doleyres et al. [11] reported that the maximum 3-HPA level (235 mM) was obtained using 400 mM glycerol, 1.6 × 10 10 CFU/mL resting cells, incubation temperatures of 30 • C and an incubation time of 45 min in the glycerol-water solution. These parameters of optimal 3-HPA production are inconsistent and likely strain-dependent.
The challenge of 3-HPA production with glycerol is the toxicity of 3-HPA accumulated to L. reuteri. To solve this problem, bisulfite can be used to entrap 3-HPA to form a non-toxic 3-HPA-bisulfite complex that can markedly increase glycerol conversion by the resting cells. The produced 3-HPA can then be released from the complex by an ion exchange resin [39].

Antimicrobial Mechanisms
Even though the antimicrobial mechanism of the reuterin system remains unclear and controversial, the structures of some antimicrobial compounds in the system have been analyzed and their antimicrobial mechanisms are initially proposed ( Figure 3). The 3-HPA dimer may exhibit antimicrobial activity through acting as a competitive inhibitor of the ribonucleotide reductase that is responsible for DNA synthesis of cells [33]. The antimicrobial activity of 3-HPA may be attributed to the aldehyde groups of 3-HPA, which can induce oxidative damages to bacteria by depletion of free sulfhydryl groups in small proteins and other molecules such as reduced glutathione and enzymes [9,40]. Furthermore, 3-HPA at concentrations of more than 10 µM can downregulate the virulence gene expression in Helicobacter pylori including vacA, flaA and luxS [41]. Notably, acrolein may deplete the free sulfhydryl groups of reduced glutathione and other enzymes through a Michael addition reaction and, thus, may induce oxidative damages to bacteria and contribute to the antimicrobial activity of the reuterin system [42]. In addition, 3-HP acts synergistically with acrolein in inhibiting microbial growth [30,43]. Overall, the application of the reuterin system as a food preservative still necessitates more insights into its antimicrobial mechanisms.

Stability
The stability of the reuterin system is affected by temperature, media, pH, etc. Additionally, 3-HPA is highly soluble in water, resistant to proteases, lipases, nucleases and liable to heat (100 °C for 10 min) [12,44]. The 3-HPA content in the supernatant produced by the two-step process was stable at −20 °C for at least 35 days, and then it slowly decreased. Moreover, repeated freeze-thaw cycles (−20/4 °C) caused a slow decrease in 3-HPA with every cycle [23]. In addition, the degradation of 3-HPA to acrolein in aqueous solution was greater at high temperatures (37 °C and 40 °C), while there was very little degradation at 4 °C for 4 weeks [45]. Similarly, IC-PAD analysis suggested that 3-HPA/acrolein did not interconvert at low temperature and acidic pH [42].
3-HPA is much more stable in aqueous solution than that in milk or MRS media [46]. After 1 h of treatment at 100 °C, the level of 3-HPA in MRS produced by the one-step fermentation process significantly reduced, but was not notably affected in the glycerolwater solution produced by the two-step fermentation process [47]. After stirred yogurt was made from raw milk containing purified 3-HPA, 3-HPA level decreased by more than 90% during 15 days of storage at 21 °C, but the reduction was slower at 4 °C. This is likely due to the fact that 3-HPA can form Schiff bases with the proteins in milk [48]. Some examples concerning stability of the reuterin system are summarized in Table 1.

Stability
The stability of the reuterin system is affected by temperature, media, pH, etc. Additionally, 3-HPA is highly soluble in water, resistant to proteases, lipases, nucleases and liable to heat (100 • C for 10 min) [12,44]. The 3-HPA content in the supernatant produced by the two-step process was stable at −20 • C for at least 35 days, and then it slowly decreased. Moreover, repeated freeze-thaw cycles (−20/4 • C) caused a slow decrease in 3-HPA with every cycle [23]. In addition, the degradation of 3-HPA to acrolein in aqueous solution was greater at high temperatures (37 • C and 40 • C), while there was very little degradation at 4 • C for 4 weeks [45]. Similarly, IC-PAD analysis suggested that 3-HPA/acrolein did not interconvert at low temperature and acidic pH [42].
3-HPA is much more stable in aqueous solution than that in milk or MRS media [46]. After 1 h of treatment at 100 • C, the level of 3-HPA in MRS produced by the one-step fermentation process significantly reduced, but was not notably affected in the glycerolwater solution produced by the two-step fermentation process [47]. After stirred yogurt was made from raw milk containing purified 3-HPA, 3-HPA level decreased by more than 90% during 15 days of storage at 21 • C, but the reduction was slower at 4 • C. This is likely due to the fact that 3-HPA can form Schiff bases with the proteins in milk [48]. Some examples concerning stability of the reuterin system are summarized in Table 1.

Toxicity of the Reuterin System
The impact of the reuterin system on human health is critical to its application in the food and medical industries. In the cytotoxicity assay using the human hepatoma cell line HepG2, the purified 3-HPA was much less toxic than acrolein and only four times more toxic than the generally recognized as safe (GRAS) flavoring diacetyl, implying a low possibility of reuterin to cause in vivo disturbance [50]. In the assay using in vitro models of the gastrointestinal tract, the 3-HPA did not affect the viability or the membrane integrity of Caco-2 cells at less than 1080 mM. Furthermore, 3-HPA had no hemolytic effect on blood cells at 270 mM [51]. In addition, the purified 3-HPA at bactericidal concentrations of 40 mg/L did not exert any skin toxicity [52].
Acrolein (2-propenal) is the main toxicant in the reuterin system, and is easily converted from 3-HPA at moderate temperature. Acrolein is commonly found in foods, and also found in the human body from endogenous production and/or dietary routes [53]. The toxicity of acrolein alone has been well-studied. The World Health Organization has classified acrolein as a class 2A carcinogen, and established a provisional tolerable oral acrolein intake of 7.5 µg kg −1 bw day −1 for non-neoplastic effects [54]. Acrolein produced by GDH-competent intestinal bacteria by metabolizing glycerol can lead to the formation of the acrolein-DNA adduct, which may induce mutations in a model system containing resting cells of L. reuteri in combination with calf thymus DNA or bovine serum albumin. However, the concentration of glycerol needed to cause the acrolein-DNA adduct-mediated mutation was much higher than the physiological concentration [55]. Nevertheless, the association between the carcinogenicity of acrolein observed in cytotoxicity assays and human health remains hypothetical, and has not been verified in clinic trials yet [56]. Although the reuterin system appears to be safe based on the results from short-term toxicity tests, the dynamic nature and complexity of the system are worthy of more attention. Therefore, studies on the dynamic changes of the reuterin system and long-term toxicity evaluation are necessary for the safety use of reuterin.

Food Preservative
The reuterin system has great potential as a food preservative due to its strong antimicrobial activity in various types of foods and high stability under different food-processing conditions. Reuterin can be used as a food preservative to extend the shelf life using the following approaches: (i) addition of 3-HPA-producing Lactobacillus and food-grade glycerol to the food to achieve in situ production of reuterin compounds, which does not require regulatory approval or label declarations [57], (ii) addition of purified, semi-purified or other forms of 3-HPA to the food, (iii) addition of 3-HPA to the food, followed by treatment with other antimicrobial hurdles.

Dairy Products
The antimicrobial activity of the reuterin system has been validated in milk, yogurt and cheese ( Table 2). Addition of L. reuteri and glycerol (50 mM) in milk, cheese and yogurt can produce sufficient 3-HPA to exert antimicrobial activity during manufacturing and storage [58]. Remarkably, addition of high concentrations of glycerol at more than 200 mM increased the redness of cheese, which may be attributed to the reaction of the excess glycerol with cheese components [59]. In situ production of 3-HPA prevented the late blowing defect caused by Clostridium tyrobutyricum in semi-hard ewe milk cheese [60,61]. Either in situ-produced or purified 3-HPA can exert antimicrobial effects in yogurt. Increased yellowish color was observed in the yogurt with 3-HPA at concentrations over 69 mM [17,62]. Nisin, lacticin 481 and enterocin AS-48 acted synergistically with 3-HPA in inactivating Staphylococcus aureus in milk, but this synergistic effect was not observed for Escherichia coli O157:H7, Salmonella enterica, Yersinia enterocolitica, Aeromonas hydrophila and Campylobacter jejuni [19]. These studies suggest that the reuterin system can be applied as a preservative in dairy products.

. Meat and Fish Products
Foodborne pathogens pose a serious threat to human health, and are easily transmitted to humans through contaminated meat products, especially raw or undercooked meat [71]. The use of the reuterin system can inactivate various foodborne pathogens in meat and meat products ( Table 3). The reuterin system produced by L. reuteri completely killed E. coli O157:H7 by at least 3.0 log CFU/g in ground beef at 4 • C at day 20 [72]. The use of L. reuteri and glycerol as meat starter cultures reduced the cell counts of E. coli O157:H7 by at least 1.0 log CFU/g during fermentation [20]. In situ-produced 3-HPA also reduced Campylobacter isolated from chicken meat [31]. The combination reduced TVC and slightly affected color, texture and volatile compounds, but was worse than lactoperoxidase system + HHP over 35 days at 4 and 10 • C. [77] Purified 3-HPA inhibited the growth of L. monocytogenes on the surface of cooked pork and sausages at refrigerated conditions without any alteration in meat quality [78,79]. Single or combined use of purified 3-HPA and high hydrostatic pressure (HHP) at 450 MPa for 5 min inhibited the growth of L. monocytogenes in cold-smoked salmon [80,81]. These studies suggest that the reuterin system is able to improve the quality of meat and fish products.

Other Products
Incorporation of 3-HPA in pectin-based edible coatings significantly reduced viable Penicillium spp. on strawberries by 2.0 log CFU/g without quality deterioration over 31 days of cold storage [82]. Washing fresh-cut lettuce with the reuterin solution containing 7.2-21.9 mM acrolein at 4 • C reduced Enterobacteriaceae, yeasts and molds by 2.1-2.8 log CFU/g and 1.3-2.0 log CFU/g, respectively. It needs to be noted that this treatment resulted in the discoloration of lettuce [83]. The undesirable discoloring effect of the reuterin system on vegetables is dose-dependent and limits the use of the reuterin system in fresh produce. The reuterin system is also not suitable as a preservative in bakery foods such as bread, because the baking process may eliminate its antimicrobial activity [47].

Health-Promoting Effects
Despite a lack of clinical trails, several in vitro and in vivo studies demonstrate that the reuterin system exhibits certain health-promoting effects including anti-infection, antiinflammation and anti-cancer activities, presumably through interacting with host microbiota and through alterations in metabolome.

Anti-Infective and Anti-Inflammatory Effect
Co-delivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas the single use of glycerol or L. reuteri Foods 2022, 11, 4000 9 of 16 was ineffective. A global unbiased microbiome and metabolomics analysis confirmed that glycerol precursor delivery with L. reuteri altered the composition and function of the human microbial community that preferentially targeted C. difficile outgrowth and toxicity [84]. In a novel in vitro colonic fermentation model where adult immobilized fecal microbiota were treated with reuterin, the addition of glycerol alone or in combination with L. reuteri increased numbers of the Lactobacillus-Enterococcus group and decreased E. coli without affecting other bacterial populations. The selective decrease in E. coli populations may be due to in situ reuterin production. These findings indicate that the reuterin system can prevent intestinal infection [85]. Moreover, the effectiveness of 3-HPA produced by L. reuteri in protection against intestinal Salmonella infections was also validated using a three-dimensional organotypic model of human colonic epithelium [86].
The frequent emergence of antibiotic-resistant pathogens has become a major threat to public health worldwide. Presently, there is no report on the resistance of pathogenic microorganisms to the reuterin system. Reuterin was considered to have a bactericidal effect on methicillin-resistant S. aureus [13]. Bennett et al. [14] reported that reuterin was the most effective against multidrug-resistant bacteria causing bovine mastitis, compared with nisin, bactofein and pediocin. The reuterin system may be a new alternative to reduce the emergence of resistant organisms and treat infections caused by resistant organisms.
Recently, Bell et al. [89] reported that 3-HPA produced by L. reuteri in the healthy intestinal microbiome inhibited colon tumorigenesis, decreased tumor growth and prolonged survival in mice by inducing protein oxidation and inhibiting ribosomal biogenesis in vivo and in vitro.

Fixation and Sanitization of Biological Tissues
3-HPA is more bactericidal but less cytotoxic than the most commonly used sterilant and crosslinker glutaraldehyde for biological tissues [90,91]. Although the rate of tissue fixation by 3-HPA is slower than that by glutaraldehyde, 3-HPA-fixed tissue has a reduced inflammatory reaction and comparable tensile strengths, calcification and resistance against degradation and free-amino group content [92,93]. Additionally, reuterin-polymerized pyridoxylated hemoglobin (RR-PLP-Hb) solution is a new option in the development of blood substitutes, because the solution has a lower oxygen affinity and longer vascular retention time than the unmodified Hb solution [94].

Other Bioactivities
Ingestion of reuterin may also exhibit other health promoting effects. Oral administration of the reuterin supernatant altered the fecal microbiome, decreased heptane and increased 3-methylbutanal in the feces of mice, suggesting the high potential of the reuterin system to relieve some diseases caused by the dysbiosis of intestinal flora [23]. Oral treatment of broilers with water supplemented with a combination of 5 mM 3-HPA and 0.08 mM microcin J25 promoted growth performance and improved health, presumably through alterations in the cecal microbiome and metabolome, indicating the potential of 3-HPA as a growth promoter instead of as an antibiotic in animals [95]. Das et al. [96] found that 3-HPA effectively ameliorated iron overload diseases via suppressing hypoxiainducible factor 2α (HIF-2α), a master transcription factor of intestinal iron absorption, using a high-throughput screen of gut microbial metabolites. More examples concerning health-promoting effects of the reuterin system are given in Table 4. [102] Trypanocidal agent The supernatant produced by the 2-step method

In vitro
The growth of culture form and motility, viability and macromolecular synthesis of culture and bloodstream forms of Trypanosoma brucei. [103] Trypanocidal agent The supernatant produced by the 2-step method and purified 3-HPA In vivo (7-week old female CF-I mice) Toxicity; levels of parasitemia; survival of the mice. [104] Anti-necrotizing enterocolitis effect The supernatant produced by the 2-step method In vivo (SD rat pups) NEC incidence; histology. [

Conclusions and Future Perspectives
The reuterin system produced by L. reuteri is a multi-component dynamic equilibrium system that was discovered nearly forty years ago. The system has powerful broadspectrum anti-microbial actions. Many factors can affect the composition of the system, such as temperature, pH and other chemical compositions. Due to the dynamic nature and lack of a fast, effective real-time assay, the contributions of specific substances in the system to the antimicrobial activity and modes of action have not been fully clarified. Thus, it is non-scientific to use only 3-HPA to represent the effective concentration of the system in most research. If the above problems are not solved, it will be difficult to achieve standardized production of commercial products of the reuterin system.
Numerous studies have shown the great potential of the reuterin system as a food preservative. However, most studies have only focused on the antibacterial effect and neglected its interaction with food components. For instance, there is a need to further investigate the reasons why the reuterin system tends to cause color changes in dairy products. Moreover, there are few research reports on the application of the system for vegetables, fruits and agriculture-based food products. Although the reuterin system has shown many health-promoting effects, in vivo and clinical studies are still lacking.
Conclusive toxicological data are the key to practical applications of the reuterin system in food and medicinal fields. The fate of the reuterin system from different sources should be fully evaluated, such as in situ-produced and purified 3-HPA. It is necessary to understand the metabolism and biotransformation pathways of the system in the body, including the skin, oral cavity, gastrointestinal tract, etc. Acute, subacute and long-term exposures of the system should be performed to obtain the safe dose range for various applications.
This review presents several insights into the dynamic nature of the reuterin system and provides critical examination on the use of the reuterin system as a food preservative or health promoting agent. Further research of the reuterin system should be carried out regarding production, antimicrobial mechanism, toxicity, bioactivities and its interactions with other food components. In addition, the efficacy of the reuterin system as a food preservative or health-boosting agent still needs to be validated in large-scale industries.

Conflicts of Interest:
The authors declare no conflict of interest.