Introduction

Advances in molecular biology can be credited with the discovery of the Weissella genus. Collins et al. [1] reported that strains with very similar phenotypic characteristics that had previously been included in the Leuconostoc genus exhibited different DNA profiles from other bacteria in the group. The authors used 16S rRNA sequences to study Weissella. They found that certain strains demonstrated specific features that were phylogenetically closer to Leuconostoc paramesenteroides than others. Collins et al. [1] recommended that L. paramesenteroides, a new species isolated from fermented sausages, and certain heterofermentative lactobacilli be classified as a new genus, Weissella.

The Weissella genus belongs to the Firmicutes phylum, Bacilli class, the Lactobacillales order, and the Leuconostocaceae family. Weissella bacteria are closely related to other lactic acid bacteria (LAB) genera. Fusco et al. [2] reported significant difficulty in separating the Weissella genus from other LAB, particularly from Leuconostoc species. Weissella species morphology may differ within the genus. The short rods with rounded to tapered ends or coccoid shapes that typify Weissella species are similar to Leuconostoc, Oenococcus, and streptococci [1]. Nevertheless, some Weissella species can present pleomorphisms under stress conditions. Cells can be present alone, in pairs, or even in small chains [1, 3]. Currently, 25 Weissella species have been determined: W. viridescens, [4], W. paramesenteroides [5], W. confusa [1], W. kandleri [6], W. halotolerans [7], W. minor [7], W. hellenica [1], W. thailandensis [8], W. soli [9], W. cibaria [10], W. koreensis[11], W. ghanensis [12], W. beninenses [13], W. fabaria [14], W. ceti [15], W. fabalis [16], W. oryzae[17], W. diestrammenae [18], W. uvarum [19], W. cryptocerci [20], W. bombi [21], W. jogaejeotgali [22], W. kimchi [23], W. muntiaci [24], and W. sagaensis [25]. A clear, well-documented summary of Weissella taxonomy and ecology can be found in Fusco et al. [2]. Although Weissella remains a relatively new genus compared with other LAB, it has been the focus of research in the past 5 years and has attracted a strong interest from the food industry. Specific Weissella strains have been studied for their ability to produce (i) antimicrobial compounds like bacteriocins and hydrogen peroxide and (ii) exopolysaccharides (EPSs) and diacetyl. The latter are of major importance to dairy applications [26,27,28,29]. Different strains have been isolated from artisanally produced foods, which demonstrate the contributing role Weissella plays in the foods’ characteristic features. Other studies have highlighted Weissella strains’ ability to survive in the gastrointestinal tract (GIT) and even grow in the gut. Both of these attributes suggest the use of Weissella species as beneficial applied probiotics [30,31,32].

The purpose of this review is to provide an overview of the Weissella genus and present the latest research and findings from the past 5 years. The paper presents the most recent techniques and approaches used in Weissella identification and characterization. It also covers the bacteria genus’ potential health benefits and its main technological features which are of interest to the food industry.

Weissella Species Identification and Characterization

The past 5 years have seen the development of new methods for identifying Weissella species. They now make it possible to distinguish Weissella species from other closely related LAB. The polyphasic identification method and sequencing and analysis of the 16S and 23S rRNA genes are two new molecular-based identification methods that allow for rapid differentiation between Lactiplantibacillus plantarum, Pediococcus pentosaceus, and Weissella confusa [33]. Complete genome sequence analysis using bioinformatic analysis can be used to describe metabolic routes and to predict potential uses for the identified strains [34,35,36,37,38]. These methods are the foundation of modern microbial taxonomy, as are matrix-assisted laser desorption ionization-time of flight method (MALDI-TOF) and database mass spectrometry [39, 40]. One recent study has shown that the colony morphology of W. confusa can change according to the adopted culture media and incubation conditions. W. confusa’s morphology can vary from rod-shaped coccobacilli—with characteristic, small, cream-colored colonies gathered in single, short or long chains—to a large, irregularly shaped, transparent morphology that lacks characteristic chain cell organization [41]. Weissella strains can also thrive in high-sugar niches and produce large amounts of dextran [41]. Weissella strains’ behavior in different substrates is key to differentiating them from other LAB.

Genomic and metatranscriptomic methods have been used to assess the metabolic and fermentative traits of W. koreensis during the kimchi fermentation process. This has led to the characterization of metabolic pathways for certain carbohydrates fermented by W. koreensis. These include D-glucose, D-mannose, D-lactose, L-malate, D-xylose, L-arabinose, D-ribose, N-acetyl-glucosamine, and gluconate [42]. The determination of carbohydrate metabolic pathways makes it possible to predict how specific Weissella substrates will ferment, as well as pinpoint those which promote higher cell multiplication rates. It also allows for an estimation in type and quantity of products generated by the fermentation process. With this information, it is possible to predict the ecosystems where Weissella will survive and thus potential food and pharmaceutical applications for specific species.

Rizzello et al. [43] delved into Weissella genus metabolism by focusing on W. cibaria and W. confusa strains. The resulting data showed that W. cibaria and W. confusa. were able to both produce phytase and use phytic acid, thus counteracting an anti-nutritional factor in legumes. The two strains were also able to reduce the raffinose oligosaccharide (RFO) concentrations of α-galactosides raffinose, verbascose, and stachyoserafinose. RFO concentrations represent another anti-nutritional factor in vegetables, especially fava beans. When W. cibaria and W. confusa strains undergo the formation process, RFO hydrolysis enables galactose production. [43]. Legumes such as fava beans, peas, and lentils are a good source of vegetable proteins. Their anti-nutritional factors include saponins, condensed tannins, and protease inhibitors. α-Galactosides and phytic acid [44] have been shown to inhibit them. Fermentation and its subsequent reduction of these anti-nutritional factors would allow these foods to be used as new protein sources for both humans and animals. These new protein sources could in turn be used to develop new products in the food industry.

Many Weissella strains have been isolated from fermented products such as kimchi, pozol, jeotgal, and ogi [33, 39, 42, 45]. These strains have been characterized in terms of the carbon source they use and the compounds they can produce. While studying carbon source metabolism, López-Hernández et al. [45] observed that strains W. cibaria, W. confusa, and W. paramesenteroides were able to metabolize D-xylose, glucose, D-fructose, D-mannose, sucrose, and D-maltose. Some strains were also able to use ribose and esculin ferric citrate. Others were able to use galactose, metabolize cellobiose, and demonstrate β-glucosidase and β-galactosidase activity. The latter is a desirable characteristic in probiotic microorganisms, as β-glucosidase breaks down certain compounds and makes them easier for the body to absorb. Anthocyanins, which have antioxidant and anti-inflammatory properties, are among these compounds [46].

New identification methods have allowed for rapid and accurate identification of Weissella strains and reduced the risk of misidentification. Characterizing Weissella strains based on their metabolic pathways opens up the possibilities for their use in the medical and food production sectors.

Beneficial Properties of Weissella

Once the metabolic pathways of Weissella have been characterized, isolated strains can be studied for use in the pharmaceutical and food industries. Weissella isolates have beneficial, probiotic features and are able to produce EPS. These make them an important bacteria in both pharmaceutical and food production sectors. Abriouel et al. [47] described the technological, functional, and pathogenic potential of Weissella genus applications in the food industry, but their review only covered research carried out before 2015. Below, our review presents and discusses the primary benefits of Weissella based on these areas of study.

Health Benefits of Weissella

Few studies in the past 5 years have examined the effects of Weissella strains on human health [48,49,50]. Some research has evaluated their effects on mice [51,52,53,54,55], gerbils [56], and beagles [57]. Most studies have demonstrated how Weissella’s probiotic characteristics and antimicrobial properties may offer health benefits. Only two strains of W. cibaria have been studied for their probiotic potential for humans. W. cibaria JW15 was studied because of how it affects cytokine and immunoglobulin natural killer cell activity, and W. cibaria CMU has been the subject of research because of its oral epithelial cell adhesion and oral colonization; W. cibaria CMU’s antibacterial activity against Fusobacterium nucleatum and Streptococcus mutans [48,49,50] represents another important species feature. A recent study found that immune functions were enhanced by an increase in natural killer cell activity for subjects who consumed probiotic W. cibaria capsules [50]. Moreover, oral ingestion of W. cibaria CMU can help to reduce halitosis and microbiota numbers in the gingival sulcus, thus improving oral health [48, 49].

Fonseca et al. [56] tested strains of Bifidobacterium longum and W. paramesenteroides to assess their probiotic potential and their impact on parasite load in gerbils. They observed the positive effect these two strains had in reducing the parasitic load of animals infected with Giardia. Strains of W. cibaria have been studied as a way to reduce cancer treatment drug side effects in mice [55]. W. cibaria strains were shown to assist in the recovery of lymphocytes, hemoglobins, and platelet levels that drop when cyclosporamide (an anti-cancer agent) is administered. Cyclosporamide is widely used in the treatment of acute and chronic leukemia, lymphoma, and a number of autoimmune diseases [55]. W. cibaria strains also proved to be efficient in treating atopic dermatitis when administered orally to mice. W. cibaria improved the clinical symptoms of lesions, such as erythema/hemorrhage, edema/excoriation, erosion, scarring/dryness, and lichenification [54]. In yet another study, W. cibaria was shown to reduce the destruction of periodontal tissue in mice and also protected against alveolar bone destruction in mice. W. cibaria co-aggregates with periodontal bacteria to produce hydrogen peroxide and bacteriocins that inhibit the production of pro-inflammatory cytokines [58]. Although clinical studies using mice are an effective research technique, these studies cannot reliably predict the outcome of human studies [59]. Further study is needed to determine the health benefits of Weissella strains for humans.

Although there have been few human or animal studies using Weissella strains in vitro research indicates a potential for future use as antimicrobial agents (Table 1) and probiotics (Table 2). Further study on animals and/or humans is necessary to prove their efficiency as a functional species. The W. confusa DD_A7 strain has been shown to be a natural alternative prophylactic agent against multidrug-resistant (MDR) ESBL (extended-spectrum β-lactamase) positive E. coli bacteria [27, 29]. W. confusa has also been shown to be effective destroying Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella Typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), and E. coli O157: H7 (ATCC 43889) pathogens. Several studies have shown that the supernatant from of W. confusa culture weakened the membrane of the pathogen E. coli O157: H7 (ATCC 43889) and damaged the integrity of its DNA [27, 29]. Another study which paired W. viridescens with high-pressure processing showed strong results when it came to protecting ready-to-eat salads from L. monocytogenes. The research showed that high pressure applied to the weakened pathogen membranes which then facilitated the antimicrobial activity produced by the W. viridescens strain [60].

Table 1 Weissella species with antimicrobial effects and the respective pathogens that each one inhibits
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Table 2 In vitro and in vivo evaluation of Weissella species as a probiotic candidate
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Recent in vitro studies have shown that W. cibaria, W. koreensis, W. confusa, and W. paramesenteroides strains are strong candidates for probiotic applications [30, 32, 61,62,63,64]. The survival of these strains in the GIT and their tolerance levels when in contact with bile and acids are important factors for intestinal colonization and pathogen protection. W. paramesenteroides has also been shown to form a biofilm which facilitates mucin adherence. It uses fructooligosaccharides (FOS) and galactooligosaccharides (GOS) as prebiotic fibers to the form of compounds that can survive the gut microbiome. W. paramesenteroides has also demonstrated antimicrobial activity against E. coli and S. aureus [62]. In another study, Anandharaj et al. [30] demonstrated that Weissella species may help reduce cholesterol due to its ability to use cholesterol from de Man, Rogosa and Sharpe (MRS) agar with added Oxgall and water-soluble cholesterol. Cholesterol reduction is common in probiotic microorganisms isolated from the intestine [65,66,67]. Interestingly, Kim et al. [58] found that using the W. cibaria D30 probiotic strain in fermented Inula britannica created a product with improved anti-inflammatory capacity.

W. confusa and W. cibaria—isolated from Plaa Som Fug—have also been shown to produce folate. Folate, or Vitamin B9, plays an important role in DNA replication, repair, and methylation. It can also act as an antioxidant [68]. Folate is important for immune and nervous system functions because it plays an integral part in the synthesis of neurotransmitters in the central nervous system [69]. This is especially the case during the early stages of uterine growth and development.

Extensive research has been carried out on Weissella genus antimicrobial activity and its potential use of strains as probiotics. However, other beneficial compounds produced by Weissella, including vitamins, have only been touched on in research thus far.

EPS Production by Weissella

EPSs are extracellular macromolecules that have potential applications in the food, medical, and pharmaceutical industries. It has been shown that Weissella strains can produce these molecules, and EPS production has been the focus of several Weissella studies (Table 3). EPS production has been primarily documented for W. confusa and W. cibaria strains, with dextran as the dominant EPS produced by the strains. Structurally, dextran molecules are highly linear. They are formed by α-1,6-linked glucose residues with branches linked to α-1,2, α-1,3, or α-1,4. The α-1,2 bonds are representative of prebiotic properties because they are resistant to adverse GIT conditions and can help to modulate the intestinal microbiota [41, 70,71,72]. EPS produced by LAB are generally recognized as safe (GRAS), which means they can be used in foods, prebiotics, and medical applications.

Table 3 Weissella species and its EPS products
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Of the LAB Weissella species, W. cibaria and W. confusa are the most important EPS producers, which is why research has focused on them and their production, purification, and characterization [41, 71,72,73,74,75,76]. Some studies have proposed ways to optimize EPS production and to test different parameters, such as the effects of different temperatures and substrates. Hu and Gänzel [71] showed that W. cibaria strains grew faster at 30 °C, but oligosaccharide production was higher at temperatures at or below 20 °C. The greatest dextran production occurred at 15 °C.

The physicochemical properties of bacteria-produced EPS are an important factor for functionality and use. Devi et al. [73] carried out physicochemical characterizations of galactan EPS produced by a W. confusa strain. They showed that the EPS displayed a high oil absorption capacity, strong emulsifying activity, and an emulsion kinetic stability of up to 15 days. These results supported the theory that galactan EPS may be a good candidate as an emulsifier in forming long-term colloidal systems in food, pharmaceutical, and cosmetic products. Other studies have shown that several Weissella strains use a dextransucrase enzyme to produce dextran. This enzyme has been the focus of isolation and purification studies for the production of EPS [77, 78] and caffeic acids [79]. Dextransucrase produced by a W. confusa strain has been shown to synthesize a rare oligosaccharide called isomelezitose. This oligosaccharide has a potent nutraceutical effect due to its ability to promote bifidobacteria growth in the colon [78].

The EPS produced by LAB have antifungal [80], anti-inflammatory, and immunomodulatory properties [51] that may be of great interest to the medical sector. EPS can also act as antioxidants [26, 28, 51, 76] and help probiotics survive passage through the GIT [81]. The linear EPS galactan produced by the W. confusa strain shows strong potential for encapsulation technology that is used to deliver bioactive compounds, probiotics, and drugs. The EPS produced by a W. confusa strain was shown to inhibit the multiplication of Candida albicans and was able to destroy up to 70% of the biofilm formed by this pathogen [80]. This suggests that it could be a promising candidate for use in antifungal treatments. Another recent study showed that W. confusa can produce mannan, an EPS made up of only monomeric units of mannose. This EPS displayed antibiofilm activity against pathogenic bacteria such as S. aureus, L. monocytogenes, S. enterica, and S. typhi. It also demonstrated potential as an antioxidant compound [61].

EPS may also have prebiotic properties. This is the case for the EPS produced by the W. cibaria strain isolated from pomelos (Citrus maximum) grown in India [82]. To be categorized as a prebiotic compound, the EPS carbohydrates must demonstrate higher metabolization levels from probiotic bacteria than from enteric bacteria present in the intestine. Dextran produced by W. cibaria was shown to stimulate the growth of L. plantarum, L. acidophilus, B. animalis, B. bifidum, and B. infantis more than E. coli and E. aerogenes [82]. This activity confirmed its classification as a prebiotic compound.

In the food industry, EPS can be used to improve the rheological characteristics and texture of fermented products. EPS can also play a role as an emulsifier and stabilizer [28, 74, 81]. EPS-producing LABs can be used as starter or secondary cultures in the production of fermented products such as yogurt [83]. The use of these cultures leads to the production of EPS in situ, where they act as natural thickeners and can eliminate the need for artificial additives [76]. Weissella strains have been used to ferment quinoa-based yogurt [84] and fruit juices [85, 86]. The EPS produced can improve the sensory characteristics of such products. One example: Orange juice concentrate was used to produce oligosaccharides. Then, after a fermentation process involving the sucrose present in the juice itself, a drink was obtained with reduced a sugar content and specific acidity and sweetness levels [85, 86].

EPS are also of interest to the frozen dough industry, as they can modify the texture and improve the end product characteristics of frozen and thawed doughs. Studies have shown that dextrans produced by different microorganisms have different functionalities. For instance, dextran produced by the W. cibaria strain demonstrated a greater ability to reinforce gelling properties compared with dextran produced by L. pseudomesenteroides when the strains were mixed with fava bean (Vicia faba L.) protein isolate [87, 88]. Weissella EPS has also been shown to improve the characteristics of wheat gluten during dough freezing and thawing. In general, the freezing-thawing process leads to an increase in water loss, a decrease in gluten viscoelasticity, and changes in water mobility and distribution. During freezing and thawing, the continuous structure of gluten is destroyed by mechanical damage caused when ice recrystallizes. An addition of W. confuse-produced EPS to doughs helped the gluten to maintain its qualities and structural integrity during the freezing-thawing process. This makes Weissella EPS promising cryoprotectant candidates for the frozen dough industry [86].

Weissella as an Emerging Pathogen

Weissella isolates with potential health benefits must also be assessed for any adverse effects. Testing is standard procedure for isolates to be used as food supplements and starter cultures to determine if they harbor virulent or antibiotic-resistant genes, even when the negative effects are not active or apparent. [89]. If or when any noxious genetic properties are present in the isolate genome, they represent a health risk because they may be transferred to other bacteria present in the food matrix or gut [90,91,92].

Although many LAB cultures found in foods and are considered safe, the biosafety of new cultures must be established before they can be used in food production [93]. Most of the studies carried out to determine Weissella strains’ probiotic potential also evaluate the strains’ health risks. These assessments include tests for hemolytic capacity and resistance to antibiotics, PCR detection of virulence genes, and biogenic amine production [94,95,96]. Kang et al. [97] assessed the safety of W. cibaria CMU and CMS1 when used for oral care probiotics. Their results confirmed the strain is resistant to vancomycin and kanamycin (antibiotics) and that the resistance is intrinsic to the genus. They also demonstrated that the antibiotic resistance of W. cibaria CMU was not transferred to recipient strains. This was confirmed by the fact that no homology for a conjugative transposon integron-specific gene was present, i.e., there was no trace of antibiotic resistance genes in either chromosomal or plasmid DNA samples. They found that the strain had negative results for β-hemolysis, mucin degradation, and platelet aggregation tests. The strain also tested negative for various toxic secondary metabolites [97].

Until now, Weissella strains have only been associated with “weissellosis,” a disease that affects rainbow trout (Oncorhynchus mykiss). The disease leads to the septicemia the fish and can cause important economic losses on fish farms. “Weissellosis” is caused by a specific species, the W. ceti [98,99,100]. Because certain Weissella strains are resistant to antibiotics such as vancomycin, teicoplanin, ceftazidime, and sulfamethoxazole, human infections remain a possibility. These would primarily affect immunocompromised patients [3, 101]. It should be noted, however, that even generally recognized probiotic strains may cause health complications. Lacticaseibacillus rhamnosus GG and L. helveticus are two examples of this [102, 103].

Even though there are few cases of this genus causing disease in humans or animals, recent studies have evaluated the safety of Weissella use in foods. Any strain considered for use in fermented foods must be approved by government regulatory commissions. Although Brazil, the USA, and European Union countries do not have specific legislation for the production of new types of yeast, they do have recommendations. In the USA, for example, some cultures are classified as safe and suitable for human consumption while others are classified as GRAS and must apply for approval from the FDA (Food and Drug Administration) [104].

In 2011, some Weissella species, including W. confusa, were added to the International Dairy Federation (IDF) inventory, a list of microorganisms allowed in food fermentation processes. The German Committee for Biological Agents has also placed W. confusa in Risk Group 1 for microorganism that are unlikely to cause human diseases [105]. W. confusa’s important technological potential as a source of probiotics and other health benefits have led to two studies that address the microorganism’s safety [105, 106]. Cupi, Elvig-Jørgensen [106] carried out a series of toxicity tests which included genotoxicity, skin irritation, eye irritation, and sub-chronic oral toxicity in rats. They found that the W. confusa strain did not show any toxic effects and therefore can be used as a safe, direct-feed microbial product for animals. Kang et al. [97] showed that W. cibaria strains could be granted GRAS status in the future because: (i) they do not have antibiotic-resistant genes, (ii) they lack antibiotic resistance transferability, (iii) their genomic sequences do not include virulent genes related to pathogenic bacteria, and (iv) they tested negative for most virulent factors (β-hemolysis, mucin degradation, etc.) and for toxic metabolic production (ammonia production, β-glucuronidase activity, etc.).

Although many studies have demonstrated the safety of Weissella, the genus has not yet been included in the Qualified Presumption of Safety (QPS) list published by the European Food Safety Authority (EFSA) [107]. Research on W. cibaria and W. confusa has been carried out to this end [96]. To be granted the QPS status, a microorganism must meet the following criteria: (i) its taxonomic identity must be well defined, (ii) the available body of knowledge must be enough to establish its safety, (iii) the lack of pathogenic properties must be established and substantiated, and (iv) its intended use must be clearly described [107].

Conclusion

Over the past 5 years, the Weissella genus has been the focus of many studies due to its strong potential as a probiotic that could be used in both the food and pharmaceutical industries. Weissella strains’ probiotic potential is attributed to its remarkable ability to survive passage through the GIT, produce antimicrobial substances for a variety of pathogens, and promote the formation of compounds that stimulate the gut microbiome. The ability of some Weissella strains to produce large amounts of EPS represents another major attribute, as EPS have prebiotic properties. Weissella EPS production also demonstrates the strains’ potential as a natural thickener for foods, among other possibilities.

Moreover, recent studies have demonstrated that Weissella strains do not have antibiotic-resistant genes and they generally test negative for virulent factors. Thus, there is a low probability that the genus could cause foodborne diseases or carry on virulent genes that may be transferred to other bacteria pathogens.