Lactobacillus exopolysaccharide: An Untapped Biopolymer

Abstract

Lactobacillus spp. belongs to a class of bacteria known as lactic acid bacteria. This classification is because they are known to produce lactic acid as a major by-product of their metabolic activities. Most Lactobacillus spp. are generally regarded as safe (GRAS) bacteria. They also produce a bio-polymeric substance known as exopolysaccharide (EPS). The EPS are popular because of their wide potential medical and industrial applications. The wide application of the EPS in medicine and industry necessitates optimal production and recovery of these polymeric substances produced by Lactobacillus spp. In this book chapter, we aim to comprehensively discuss Lactobacillus EPS, its inherent properties, potential pharmaceutical and industrial applications. We also point to its contribution towards the achievement of the 3rd and 9th components of the United Nations Sustainable Development Goals which are to establish good health and wellbeing and to promote industrialization, innovation, and infrastructure respectively.

1. Introduction

1.1 Overview of Lactobacillus as a genus

The genus Lactobacillus spp. are members of lactic acid bacteria (LAB) belonging to the family Lactobacillaceae, order Lactobacillales, class Bacilli, and phylum Firmicutes. For the past decades, the Lactobacillus genera have been revised and new genus names were assigned. From a taxonomic point of view, the genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological, and genotypic levels [1]. More than any genera of LAB, the genus Lactobacillus is generally regarded as safe (GRAS) and finds application in the food, dairy, cosmetic and pharmaceutical industries. The genus Lactobacillus is non-spore-forming, catalase-negative or pseudocatalase, oxidase negative, obligate saccharolytic rods or coccobacilli generally characterized by a low guanine and cytosine (GC) content of the genome although the upper limit of GC content reaches 59.2 mol % [2]. The availability of 16S rRNA gene sequence and genome data has ultimately unlocked the frontiers of knowledge into the evolutionary relationships of Lactobacillus species.

Based on the type of sugar fermentation pathway, lactobacilli can be categorized into three groups, (i) obligatelyhomofermentative, that produce only lactic acid from glucose as an end product of carbohydrate metabolism through the glycolysis or Embden-Meyerhof- Parnas (EMP) pathway; (ii) facultativelyheterofermentative, that produce a mixture of lactic and acetic acid, diacetyl, acetoin, and carbon dioxide as end products of carbohydrate metabolism via the glycolysis or the phosphoketolase pathway, and; (iii) obligatelyheterofermentative, that produce lactic and acetic acid, or ethanol, and CO₂ as end products of carbohydrate metabolism via the phosphoketolase (6-phosphogluconate) pathway [3].

The lactobacilli have varied resistance to different NaCl concentrations, antibiotics, and temperature or pH range mostly due to cellular fatty acids, isoprenoidquinones, and other characteristics of their cell wall composition [4].

Until recent times, most studies on lactobacilli were focused on their application in food fermentation and formed the greater percentage of probiotics in current use. However, the production of exopolysaccharides biopolymers has added a new dimension to the usefulness of lactobacilli.

This book chapter aims to critically look at Lactobacillus EPS, its inherent properties, potential pharmaceutical and industrial applications, and how to harness it in line with the 3rd and 9th components of the United Nations Sustainable Development Goals which include the establishment of good health and wellbeing and to promote industrialization, innovation, and infrastructure respectively.

2. Composition of Lactobacillus exopolysaccharide (EPS)

The bioproduction of exopolysaccharides is of universal occurrence among eukaryotic (plants, seaweeds) and prokaryotic (bacteria) organisms. Microbial exopolysaccharides have many uses in numerous fields including food industries, farming, textiles, cosmetics, bioremediation and therapeutics, and pharmacy because of their different composition, structure, physical and chemical properties [5, 6].

Based on the type of monosaccharide monomers, EPS are divided into two groups of homopolysaccharides and heteropolysaccharides. The Lactobacillus spp. like other LAB can synthesize homopolysaccharides or heteropolysaccharides. The synthesized homopolysaccharides are glucans or fructans, which are composed of only one type of monosaccharide (glucose or fructose, respectively), whereas the heteropolysaccharides contain different types of monosaccharides in different proportions [7].

The homopolysaccharides EPS of lactobacilli with a molecular weight of greater than 106 Dalton are either branched or unbranched and composed of either glucose or fructose and are categorized into α-D-glucans (Dextran, Mutan, Alternan, and Reuteran), β-D-glucans, fructans (Levan and Inulin) and polygalactans [6, 8, 9]. Similarly, heteropolysaccharides EPS of lactobacilli have a molecular weight ranging from 104 and 6.0 × 106 Dalton and are made up of common sugars (D-glucose, D-galactose), rare sugars (L-rhamnose, mannose, arabinose, xylose, fucose, N-acetylglucosamine, N-acetylgalactosamine or glucuronic acid) and non-carbohydrate components (acetate, phosphate, sulfate, pyruvate, propionate, glycerate, amino acid, L- glutamate, and succinate [6, 9, 10]. Some examples of heteropolysaccharides EPS of lactobacilli are gellan, xanthan, and kefiran [6].

3. Importance of Lactobacillus exopolysaccharides (EPS)

3.1 Health benefits of Lactobacillus exopolysaccharide (EPS)

Lactobacillus exopolysaccharides though are produced to help the bacteria withstand unfavorable environmental conditions such as desiccation, toxic materials, and osmotic stress [11], their health benefits are far-reaching. These health benefits include.

3.1.1 Lactobacillus EPS as rotavirus therapeutic agent or oral vaccine adjuvants

Several studies have shown the importance of Lactobacillus EPS in rotavirus-induced diarrhea in children. Lactobacillus has shown an ability to suppress the replication of rotavirus through the improvement of the intestinal barrier [12]. This attribute may be linked to the EPS produced by the bacteria. The study by Kim et al. [13], showed a potential rotavirus therapeutic/vaccine adjuvant effect from EPS produced by Lactobacillus plantarum. The EPS according to the murine model study caused a reduction in the duration of diarrhea, reduced lesions of the epithelium, reduced replication of the rotavirus in the intestine, and a reduction in the time of recovery of the suckling mice [13].

3.1.2 Antioxidant property

Basically, free radicals are atoms that can damage cells leading to sickness and aging. Antioxidants are substances that can reduce the reactivity of free radicals through the donation of electrons. A study by Adebayo-Tayo and Fashogbon 2020, revealed that exopolysaccharide from Lactobacillus.

delburecki subsp. bulgaricus had an antioxidant activity that is comparable to ascorbic acid [14]. Other studies by Tang et al. [15], Silva et al. [16], and Yang et al. [17] all show potential antioxidant effects of Lactobacillus exopolysaccharide [15, 16, 17]. The antioxidant potential of EPS may be due to the bioactive component in its moiety that is capable of donating hydrogen ions [18].

3.1.3 Anti-cancer activity

Cancer is an abnormal growth of cells with consequent destruction of other cells and organs leading to death. Cancerous conditions are usually serious medical conditions that require serious attention. Methods for the treatment of cancer which include the use of chemotherapeutic agents, radiation, and surgery are invasive. This is because chemotherapeutics and radiation treatments are non-selective as they destroy both normal and cancerous cells leading to serious adverse effects. Based on these explanations, emphasis is now on natural products with anti-cancer activity as they are most likely to come with minimal adverse effects when compared with other agents. The EPS of Lactobacillus has been studied for potential anticancer activity with a lot of promising results. EPS of Lactobacillus gasseri showed good anti-proliferative activity against cervical cancer cell lines [19]. EPS of L. plantarum caused an increased expression of pro-apoptotic genes in mouse intestinal epithelial cancer [20]. Lactobacillus kefri and other Lactobacillus strains have shown activity in colorectal cancer [21, 22]. The mechanisms of anti-cancer activity of these EPS are postulated to be through the improvement of immunity, prevention of tumorigenesis, and induction of cancer cell apoptosis [23].

3.1.4 Antimicrobial properties of EPS

Probiotic bacteria are known to produce antimicrobial substances such as bacteriocin, organic acids, etc. Research from several scientists has shown that Lactobacillus EPS possesses antimicrobial properties. In vitro study of the antimicrobial property of EPS from Lactobacillus rhamnosus isolated from human breast milk showed significant activity against E.coli and Salmonella typhimurium [24]. Studies by other scientists confirmed the potential antimicrobial properties of EPS from other Lactobacillus spp. [25, 26].

3.1.5 Antiviral activity

The improvement of intestinal health through the use of immunobiotics is quite trending at the moment. Immunobiotics are supplements that contain immunoglobulin combined with probiotics and prebiotics. Immunobiotics protection against viral infection is through enhancement of innate and adaptive immunity that leads to the reduction of the duration of the disease, number of episodes, and viral shedding [27]. Pattern recognition receptors through interaction with EPS allow communication between the immunobiotics and the host. EPS of Lactobacillus delbrueckii showed an improved antiviral activity [28]. L. plantarum antirotavirus activity has been mentioned earlier [13]. Anti-adenovirus activity from EPS of Lactobacillus, Leuconostoc, and Pediococcus spp. has been recorded [29]. Another Study using a Swine testicular cell line, showed an inhibitory effect on transmissible gastroenteritis coronavirus infection by EPS from L. plantarum [30].

3.1.6 Other medical importance

These include anti-inflammatory [31], anti-cholesterol [32], Immunostimulatory [33], anti-diabetic properties [34].

3.2 The importance of Lactobacillus EPS in the food and dairy industry

3.2.1 EPS as a bioflocculant

Flocculants are agents that can cause the aggregation of dispersed particles in a suspension [35]. This makes the suspended particles easy to remove thus presenting a good mouthfeel of the food product. These flocculants can be grouped into inorganic, organic, and bioflocculants [36]. The use of inorganic and organic flocculants is associated with biological toxicities [36, 37]. These biological toxicities are of serious concern and gave birth to more reliable and biologically friendly bioflocculants. The bioflocculants have the advantages of non-toxicity, biodegradability, and no residual pollution [38]. Studies on the use of Lactobacillus exopolysaccharides as a bioflocculant have been carried out with promising results [16, 39].

3.2.2 EPS as biothickner and gelling agent

Thickeners and gelling agents are usually polysaccharides or protein derivatives. They are usually added to food to increase viscosity and stability while maintaining other desired characteristics of the food product. They are used extensively in dairy and non-dairy products. The use of Lactobacillus EPS as biothickners/gelling agents is shown by the works of [1, 40, 41, 42]. Lactobacillus EPS are widely used in the dairy industry as thickeners and texturizers with a major function of stabilizing the milk and its constituents [43].

3.2.3 EPS as a meat preservative and quality enhancer

The use of lactic acid bacteria to preserve meat is an old practice that has extensively helped in the sustenance of the food industry. These bacteria carry out this preservation through the production of antibacterial substances such as acetic acid, lactic acid, and bacteriocin. They also compete with other food spoilage pathogens for available nutritional substances hence the survival of the fittest. Another wonderful attribute of Lactic acid bacteria is their ability to produce EPS. This EPS is known to protect the bacteria against environmental stress such as high salt concentration, low PH, and low water activity [44]. This protection is known to sustain the preservative ability of LAB. The EPS is also known to reduce fat in the meat. The health importance of reduced fat in meat cannot be over-emphasized. This has led to a growing demand by consumers for fat-reduced meat. A study by Hilbig et al. [45] reported that EPS matrices formed by LAB in certain German meat products were able to cause a fat reduction and improved the quality of the meat product [45].

3.2.4 Exopolysaccharide as an inhibitor of syneresis

Syneresis is the expulsion of a liquid from a gel. It has been shown to affect food and food products negatively. This phenomenon known as syneresis should be minimized in food products without affecting the inert property of the food product. Lactobacillus exopolysaccharide according to the study was able to prevent syneresis in starch [46]. Other studies by Lynch et al. [47] and Han et al. [48] gave vivid importance to the syneresis preventive ability of exopolysaccharides produced by lactic acid bacteria [47, 48]. The exopolysaccharide’s ability to prevent syneresis is because of its inert high water-binding affinity. This will then make water be retained in the food or dairy product (Table 1).

S/N	Organism	Medical/industrial applications	Referrences
1.	Lactobacillus plantarum	Cholesterol lowering property	Dilna et al. [32]
2.	Lactobacillusdelbrueckii subsp. Bulgaricus and Lactobacillus acidophilus	Improve the texture of fermented food	Vinogradov et al. [49]; Charchoghlyan et al. [50]
3.	Lactobacillus johnsonii and Lactobacillus casei	Immunomodulatory property	Gorska et al. [51]; Xiu et al. [52]
4.	Lactobacillus rhamnosus and Lactobacillus gasseri	Antimicrobial property	RiazRajoka et al. [24]; Parveen et al. [25]
5.	Lactobacillusdelbrueckiiand L. plantarum	Antiviral property	Kanmani et al. [28]; Type et al. [53]; Yang et al. [30]
6.	L. gasseri,Lactobacillus brevis, and Lactobacillus kefri	Anticancer	Sungur et al. [19]; RiazRajoka et al. [21]; RiazRajoka et al. [54]
7.	Lactobacillus paraplantarum	Anti-inflammatory property	Pecikoza et al. [55]
8.	Lactobacillus delbrueckiisubspbulgaricus	Antioxidant property	Tang et al. [15]
9.	L. casei	Viscocity enhancer in food industry	Zheng et al. [56]
10	L. plantarum	Enhancer of thr rheological property of food	Silva et al. [16]

Table 1.

The medical and industrial application of Lactobacillus EPS.

4. Challenges facing the industrial applications of EPS

4.1 Poor yield of the EPS by Lactobacillus spp.

The major challenge facing the industrial and medical applications of Lactobacillus exopolysaccharides is the poor production of the polymer by the bacteria. It is therefore very important that critical and intensified scientific experiments geared towards solving this problem be prioritized. This is because of the obvious importance of this EPS in medicine and industry. We strongly believe that the ability of the scientific community to solve this problem of poor yield rest on intensified scientific research. However, several types of research have been conducted and some are ongoing towards finding ways of improving the yield of this very important polymer [57, 58, 59, 60].

4.2 Difficulty in isolation and characterization

The methods of extraction and characterization pose serious challenges to the industrial applications of EPS. There are several methods for the extraction of EPS from LAB. However, the physico-chemical properties of this polymeric substance can be affected by the method adopted [61]. The extraction and isolation of EPS usually involve the use of organic solvents, enzymes, sugars, filtration, chromatographic procedures, dialysis, etc. [62]. In all these, the most important thing is to target a high yield of pure EPS utilizing cheaper processes. To achieve this some important aspects of the process are taken into consideration namely the removal of proteins to avoid co-precipitation with EPS, and the prevention of reaction of the EPS with the solvents and components of the medium [61]. Since it is common knowledge that the major challenge of full industrial utilization of EPS is poor yield, there is, therefore, a need to adopt the best strategy for maximal extraction and characterization of the small quantity released by the bacteria. However, this process according to Badel et al. [63] is very laborious and indeed challenging [63].

5. Ways of mitigating the problems associated with EPS production by Lactobacillus spp.

A critical look at available scientific literature has shown that the importance of Lactobacillus EPS in medicine and industries cannot be overemphasized. It is therefore important that serious research and strategies targeted at improving the yield and manipulating its structures to achieve desired properties be the focus of scientists.

5.1 Screening for high EPS yielding strains

Badel et al. [63] in their study suggested that about 165 spp. of Lactobacillus are capable of EPS production [63]. Having identified these spp., there is then a need for extensive screening of these spp. to identify strains with potentially high EPS yielding capacity. It is a known fact that EPS are produced by these organisms to help them withstand environmental stress. Based on this fact it is, therefore, possible to experimentally create some of these stress conditions in the laboratories to enhance EPS production by the potentially high yielding strains. The stress may be in the form of deprivation or excessive introduction of certain nutritional substances. Organisms are known to respond differently to these challenges. A typical example is shown with Lactobacillus lactis subsp. cremoris and Lactobacillus delbreckii subsp. bulgaricus. While nitrogen deprivation improved the yield of EPS by L. lactis subsp. cremoris [64], nitrogen enrichment caused an improved EPS production by Lactobacillus delbreckii subsp. bulgaricus [65]. Similarly, sugar stress has also been shown to affect EPS production by Lactobacillus spp. It has been established that an increase in the concentration of sucrose in various media for the growth of Lactobacillus spp. has resulted in a significant increase in EPS production by the organisms [66, 67, 68]. Other stress conditions that enhance EPS production include osmotic stress [66] and temperature stress [69]. The stress-induced by the presence of other organisms has also led to enhanced EPS production. Several studies have shown that co-cultivation of some strains of Lactobacillus spp. with Saccharomyces cerevisiae led to improved production of EPS [70, 71, 72].

5.2 Development of genetically engineered strain

The advent of biotechnology and molecular biology has led to finding solutions to certain biological and physiological problems. Genetic engineering being the modification of organisms or population of organisms through manipulation or recombination of DNA or other nucleic acid molecules has led to the discovery of important substances such as human insulin and important vaccines. Due to the increasing importance of EPS in medicine and industries, a good understanding of genes encoding EPS production and their biosynthetic pathways has become very necessary. These studies have shown that there are four pathways involved in EPS production namely wzy-dependent pathway, the ATP-binding ABC transporter pathway, the synthase-dependent pathway, and extracellular synthesis by sucrose-dependent pathway [73]. Lactobacillus spp. are known to produce EPS via the wzy-dependent pathway [74]. Genetic engineering technology can enhance EPS production through manipulation of carbon metabolism and regulation of the biosynthetic pathways for its production [75]. Genetic manipulations leading to overexpression of genes and gene knockouts have caused an increase and structural changes in the produced EPS with desired characteristics.

5.3 Exploration of efficient extraction method

The production of a high yield of EPS requires optimization of the media for the growth of the bacteria. This involves the addition of substances to the established media for the growth of the bacteria. These additional substances aimed at enhancing EPS production may pose serious problems during the recovery of the final products. Therefore, successful production can be said to rely substantially on the use of a medium that not only allow high yield but also one whose components do not interfere with EPS component, recovery, and quantification [76]. In choosing a media it is advisable to choose one whose components are defined with minimal interfering compounds. Based on the complexities (no of additional substances for enrichment) of different media the more laborious the extraction/purification of the EPS. In complex media, a lot of pre-treatments and treatments are usually carried out. It is important to state that the use of complex media for a supposed gain in yield should be weighed against the laborious processes of the final recovery. This is because the possible gain in yield could be lost in the high cost and laborious processes of recovery. At times simple media may give substantial yield without the rigorous processes of recovery involved in the use of complex media. In all, the optimal recovery of the produced EPS should be the desire of the whole process.

6. Lactobacillus EPS and the sustainable development goal 2030 agenda of the United Nations

The 2030 agenda of the United Nations sustainable development goals was birthed in 2015 as a fallout of an agreement reached by 195 countries. It is subdivided into 17 components namely (1) Elimination of Poverty (2) Erasing Hunger (3) Establishing Good Health and Well-Being (4) Provision of Quality Education (5) Enforcement of Gender Equality (6) Improving Clean Water and Sanitation (7) Growing Affordable and Clean Energy (8) Creation of Decent Work and Economic Growth (9) Increasing Industry, Innovation, and Infrastructure (10) Reduction of Inequality (11) Mobilizing Sustainable Cities and Communities (12) Influencing Responsible Consumption and Production (13) Organization of Climate Action (14) Developing Life Below Water (15) Advancement of Life On Land (16) Guarantying Peace, Justice, and Strong Institutions (17) Building Partnerships for the Goal. The 3rd and 9th component is of special interest to us as it concerns our biopolymer. The use of natural products in therapy is associated with a more patient-friendly outcome. They usually do not produce as many side effects as synthetic products [77]. The commercial availability of this biopolymer means we are to witness new pharmaceutical formulations of important agents containing the EPS. This development will establish good health and wellbeing through the use of safe and efficacious pharmaceutical products. The pharmaceutical industry will also experience an increase in new therapeutic products for various indications containing the EPS. The dairy and meat industries are not left out as the use of EPS will be made to be more popular.

7. Conclusion and future perspectives

The EPS produced by Lactobacillus spp. are important biopolymers whose potential medical, pharmaceutical and industrial applications are still underutilized. The major problem of this underutilization rest mainly on the poor yield of the biopolymer. The selection of potentially high-yielding strains, exploitation of biotechnological principles through genetic engineering, and exploration of efficient extraction processes through extensive research will surely mitigate this problem and help give the biopolymer the required boost it deserves. Scientists in the field of probiotics should make concerted efforts towards the commercialization of EPS. Here we mean making characterized and lyophilized EPS powder commercially available for medical, pharmaceutical and industrial applications, especially in the food industry. The commercial availability of a lyophilized EPS powder will be a welcome development, especially for scientists in the pharmaceutical and food industry who really understand the importance of this biopolymer.

Since EPS is a biopolymer, its use in therapeutic formulations will be more patient-friendly devoid of side effects associated with synthetic or semi-synthetic chemicals. Furthermore, the commercial availability of fully characterized biopolymeric substances of Lactobacillus origin will create a huge innovation and industrial revolution in the pharmaceutical space this is because of new formulations of anticancer, antiviral, antidiabetic, anti-inflammatory, immunostimulants, cholesterol-lowering agents, etc. will be available for therapeutic applications. The same will be applicable in the dairy and meat industries as they will witness a reduction/complete elimination of the use of synthetic polymers. The full industrial and pharmaceutical application of EPS will contribute positively towards the establishment of improved health and wellbeing, as well as promote industrialization, drive innovation and increase infrastructure as envisaged in the United Nations Sustainable Development Goals.

Conflict of interest

The authors declare that there is no conflict of interest.