A review of lactic acid bacteria isolated from marine animals: their species, isolation site and applications

Lambuk, F., *Mazlan, N., Thung, T.Y., New, C.Y., Rinai, K.R. and Son, R. Faculty of Health and Life Sciences, Management and Science University, University Drive, Off Persiaran Olahraga, Seksyen 13, 40100 Shah Alam, Selangor, Malaysia Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia. Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, 3800 Australia Go Plus Services Sdn. Bhd., 97A, Jalan BP 6/3, Bandar Bukit Puchong, 47210 Puchong, Selangor, Malaysia Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia


Introduction
Lactic acid bacteria (LAB) are microorganisms known for their benefits to mankind. They are widely distributed in carbohydrate-rich environments and typically found in decayed plants and animal matter. LAB are described as a group of Gram-positive with the shape of rods or cocci, non-spore-forming, lack of catalase activity, anaerobic or facultative aerobic, nonmotile and acid-tolerant microorganism (Florou-Paneri et al., 2013, Quinto et al., 2014, Gupta et al., 2018Bintsis, 2018). LAB can be identified based on the morphology, their ability to ferment carbohydrates, carbon dioxide production, growth in different temperature and their ability to grow at high salt concentrations. Figure 1 shows the basic structure of a Lactic Acid Bacteria, Lactobacillus. LAB are classified into six main groups: Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Pediococcus (Bennani et al., 2017).
These types of bacteria are mostly present in the human body as normal flora to the alimentary tract. Their presence ensured control over less friendly bacteria as well as giving other benefits to the host (Gupta et al., 2018). LAB have been regarded as safe food-grade microorganisms (Xu et al., 2016). They have been used as food preservatives and modifiers in flavours as well as the texture of food. LAB are also important in the fermentation of dairy products, meats, vegetables and Marine animals are adapted to live in saltwater. The animals are able to regulate salt intake, obtain oxygen and withstand the saltwater pressure (Kennedy, 2019). They live at all levels of the oceanic water column. The animals can be seen in the upper ocean, in deeper waters, and all over the ocean basins. The abundance of organisms decreases with depth (Cochran, 2014). LAB are generally considered as favourable bacteria to marine animals. They are known for their ability to act against bacterial pathogens (Merrifield et al., 2014). LAB are mostly isolated from the gastrointestinal tract (GIT) and muscle of marine animals. Variation of LAB suggests that real variation exists between marine animals' species and geographical location (Buntin et al., 2008).

Characterisation of lactic acid bacteria
Lactate and acetate are the main products produced by LAB during carbohydrates fermentation. Previous studies proved that some LAB performed as an antagonist towards pathogenic and spoilage microorganisms (Moosavi-Nasab et al., 2014). There are two metabolic categories of LAB based on the sugar fermentation patterns: homofermentative, and heterofermentative. Figure 2 shows the metabolic pathway in LAB. Homofermentative LAB transforms almost all of the sugar into lactic acid. Meanwhile, the heterofermentative LAB does not produce lactic acid as the main product of fermentation. They may produce ethanol or acetate as the by-products (Ganzle, 2015). In general, the main products of LAB fermentation include alcohol, carbon dioxide and organic acid. They also produce aromatic molecules, vitamins, or bioactive peptides (O'Bryan et al., 2015). Some LAB possesses a limited capacity to synthesize amino acids from inorganic nitrogen sources. They depend on the presence of amino acids available in the environment or medium of growth. Proteinase and peptidase can be found as extracellular or intracellular substances of the cell. A few LAB strains are able to metabolize lipids. The strains have either intracellular or extracellular lipases. Furthermore, they perform unique fatty acid transformation reactions including isomerization, saturation, hydration and dehydration. Their metabolic activities are also able to provide health benefits to the host. Lactobacilli are able to break down cholesterol into serum lipid and this had been proved by a few studies on mice, preclinical and clinical trials (Hayek et al., 2013). Some LAB can metabolize citrate. This metabolism requires citrate transportation, citrate conversion into oxaloacetate and pyruvate. Citrate metabolism by LAB leads to the production of 4-carbon compounds, i.e diacetyl, acetoin and 2,3-butanediol. These compounds possess aromatic properties and they give a certain odour to some fermented products (Cadwallader et al., 2009).

Application of lactic acid bacteria
Colonization of lactic acid bacteria is important to the development and physiology of the host. In the gut, they contribute to nutrient absorption, immune response, mucosal tolerance and epithelial development. A balanced population of intestinal bacteria is crucial in maintaining the health of the host (Rungrassamee et al., 2014, Russo et al., 2015. The intestinal tract is considered to be a valuable waste and a good source for LAB isolation (Moosavi-Nasab et al., 2014).
LAB have been applied as starter cultures in foods and beverages due to their ability to improve nutritional, organoleptic, technological and shelf-life characteristics. Lactic acid is the main product of fermentation, followed by acetic acid, while LAB can also produce ethanol, bacteriocin, aroma compounds, exopolysaccharides and some enzymes (Florou-Paneri et al., 2013, Mozzi, 2016. LAB is widely used as a source of probiotics because they play important roles in the host digestive tract, improving its immune status, modulating the bacterial community, and antagonizing opportunistic pathogens. They enhance the balance of the microbial community in the intestine, confer protection against potential pathogenic bacteria, and prevent and/or cure intestinal diseases (Azat et al., 2016). Probiotics have also been MINI REVIEW frequently administered in aquaculture because they give out several benefits including immunological, nutritional, and environmental benefits (Sha et al., 2016). LAB acts as the preservation to the food by producing metabolites such as lactic acid, fatty acid and bacteriocin by inhibiting the growth of spoilage pathogenic bacteria. The compounds produced by these LAB interact with the cell membranes of the harmful pathogens and inhibit the growth (Azat et al., 2016;Gupta et al., 2018). Intestinal LAB also can produce some vitamins that are needed by the body of the host (Florou-Paneri et al., 2013). Lactobacillus spp. (L. lactis, L. plantarum, L. bulgaricus), Streptococcus spp. and Enterococcus spp. are able to produce folate (Masuda et al., 2014).
Lactobacillus spp. has the ability to produce vitamin B12. Strains of genera Lactococcus, Lactobacillus, Enterococcus, Leuconostoc and Streptococcus are able to produce vitamin K. LAB release a variety of enzymes into the gastrointestinal tract. Enzymes produced by the LAB during food fermentation are amylases and peptidases. Amylases are being used in sourdough technology and peptidases are used for cheese making. LAB are also important in winemaking. The bacteria grow in wine during malolactic fermentation, following alcoholic fermentation. These secondary modifications improve the taste and flavour of wine (Florou-Paneri et al., 2013).

Shellfish
LAB isolated from the gastrointestinal tract of wild shrimp was revealed to have high prevalence and diversity. The LAB isolated from the wild shrimp also possesses inhibitory properties towards Vibrio harveyi. Coccoid LAB is the most prevalent LAB in the gastrointestinal tract of aquatic animals (Kongnum et al., 2012). Table 1 shows the LAB isolated from various species of shellfish. Streptococcus phocae isolated from Indian white shrimp by Kanmani and colleagues (2010) has the ability to restrain mortality and improve the survival rate of the shrimp. Lactococcus lactis isolated from kuruma shrimp (Marsupenaeus japonicus) was claimed to have the potential in controlling and preventing Vibrio penaeicida infection (Maeda et al., 2014). Samples taken from muscle and intestine of shrimp collected from Tunisia showed the presence of Lb. paracasei (Boulares et al., 2011). Microbiota that inhabits the intestine of wild-caught and domesticated giant tiger prawn (Penaeus monodon) were compared by Rungrassamee and team (2014). The study showed the presence of Lactobacillus sp., Lactococcus sp. and Pediococcus sp. in wild-caught giant tiger prawns. Out of the 24 isolates from the GIT of black tiger shrimp (Penaeus monodon) and ornate spiny lobster (Panulirus ornatus), 8.3% was detected as Enterococcus faecalis. This strain showed strong antimicrobial activity against Proteus sp., Proteus mirabilis and E. coli. However, the antimicrobial activity of its bacteriocins lost completely after incubation at 60ᵒC for 30 mins (Nguyen et al., 2014). Azahar et al (2018) had isolated 14 probiotic strains from GIT of prawn (Macrobrachium rosenbergii). Molecular identification using 16s rRNA genes sequences identified them as E. faecalis, Lc. lactis and Lc. garvieae. Three of the 14 strains showed antimicrobial activities towards V. parahaemolyticus, V. alginolyticus and Aeromonas hydrophila. However, the authors did not mention the specific strains which possess growth inhibition towards the pathogens.
Lactobacillus spp. were isolated from the oyster (Crassostrea gigas) by Lee and colleagues (2010) and was expected to be more adaptable to marine aquaculture conditions compared to freshwater animals. Kang et al. (2016) claimed that Lb. rhamnosus MH22 exhibited relatively high antagonism to the two strains of Vibrio spp. Lb. plantarum isolated from the muscle of shellfish; oyster (Crassostrea gigas) and shortnek clam (Tapes philippinarum) showed inhibitory activity against pathogens including E. coli, Edwardsiella tarda, Staphylococcus aureus, Salmonella enterica serovar Enteriditis, Streptococcus inae, S. enterica serovar Typhimurium, V. ichthyoenteri and V. parahaemolyticus (Kang et al., 2016). Meanwhile, Lactobacillus paracasei ssp. paracasei was successfully isolated from the gastrointestinal tract of abalone (Holiotis asinina). This species was discovered to be able to inhibit the growth of enteropathogenic bacteria (E. coli, Bacillus cereus and S. aureus) and able to grow in acidic conditions and tolerant of bile during 24 hrs incubation (Sarkono et al., 2010).
Furthermore, the intestinal content of clam (Meretrix lamarckii) may harbour different Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris strains as reported by Itoi et al. (2013). The clam inhabits the intertidal zone with the changes of osmotic pressure, dissolved oxygen and temperature. The changes would also force the LAB to adapt to the condition. Leuconostoc pentosus and E. faecium was isolated from the whole body without a shell of oyster (Crassostrea gigas). Enterococcus faecium was found to be resistant to 10 types of antibiotics. Probiotic strains with antibiotic resistance would be captivating if the probiotic is administered during antibiotic treatment (Kang et al., 2017). Kim and colleagues (2017)   A study conducted by Talpur and colleagues (2012) indicated that Lb. plantarum, Lb. salivarius and Lb. rhamnosus isolated from the gastrointestinal tract of blue swimming crab (Portunus pelagicus)improved the survival of P. pelagicus larvae. GIT of female mud crab (Scylla paramamosain) assembled 0.37% abundance Lactobacillus genus. Genus of Lactococcus and Lactobacillus were found in GIT of male samples with a relative abundance of 0.7% and 1.10% respectively (Wei et al., 2019). S. agalactiae was isolated from GIT of swimming crab (Callinectes sp.). The swimming crab was collected from the open lagoon at 28ᵒC and from the market samples at 0ᵒC. The study showed the least hemolytic values compared to other isolated bacteria. This strain was previously belonging to pyrogenic mastitis in cows (Uaboi-Egbenni et al., 2010).

Finfish
LAB has gained attention with respect to its beneficial effects on fish health. LAB play important role in the fish GIT. The LAB is able to stimulate the fish GI development, digestive function, mucosal tolerance, stimulate immune response and improve disease resistance (Ringo et al., 2018). Wild fish could be a substantial source of LAB that could be suitable for use as probiotics in feed for fish (Kim et al., 2013). Table 2 shows the LAB isolated from finfish. A study conducted by Salas-Leiva and colleagues (2017) reported that they had isolated Carnobacterium, C. divergen, Lactobacillus, Streptococcus, Vagococcus, and Weissella from the intestine of fine flounder (Paralichthys adspersus). Boulares and team (2011) had isolated LAB from muscle and intestine of eight types of marine fish. Lactobacillus was mostly found genus in fish followed by Lactococcus. The team also isolated Lb. paracasei from red mullet, Lb. brevaris from whiting and Leu. citream from pageot. The most dominant phylum isolated from Atlantic salmon (Salmo salar) was Firmicutes. Carnobacterium, C. divergen, Lactobacillus, Lactococcus, Streptococcus were found in the gastrointestinal tract of the fish (Dehler et al., 2017). A study conducted by Svanevik and Lunestad (2011) was to examine the microorganisms obtained from the gill, skin and GIT of Atlantic mackerel (Scomber scombrus). As the result, they found two species of LAB which were Vagococcus sp. (isolated from GIT) and V. carniphilus (isolated from gill, skin and GIT).
Lactobacillus. buchneri, Lc. lactis, Lb. acidophilus, Lb. fermentum, and S. salivarius isolated from the intestine of Narrow-barred Spanish mackerel (Scomberomorus commerson) showed different degrees of inhibitory activity. It was probably due to the synergistic effect of both bacteria and antimicrobial substances, hence enhancing their antimicrobial effect against Listeria innocua (Moosavi-Nasab et al., 2014). S. parauberis strain maris rubric was identified from broomtail wrasse (Cheilinus lunulatus). It is known for causing bovine mastitis and this type of LAB was tested to be resistant to oxytetracycline, baytril (enofloxacin) and doxycycline. Streptococcus iniae was isolated from the goatfish (Parupeneus sp.), grouper (Epinephelus fasciatus), and rabbitfish (Siganus rivulatus). This strain was tested to be sensitive to oxytetracycline, chloramphenicol, florfenicol, baytril (enrofloxacin), doxycycline, sulphamethoxazole/trimethoprim and clindamycin (Ucko et al., 2013). The LAB isolated from the gut of Indian mackerel (Rastrelliger kanagurta) were identified as Lb. plantarum, Lb. viridiscens, Lb. bulgaricus, and Lb. brevis. From the isolated LAB, Lb. plantarum was found to possess good antibacterial activity (Ghosh et al., 2014). Enterococcus durans was isolated from the viscera of five species of fish from the Mediterranean coast, Tunisia. E. faecium was the only LAB found from red mullet (Mullus surmutelus) and Lc. lactis from picarel (Spicara smaris). Lc. lactis from picarels showed a broader inhibitory spectrum and displayed important inhibitory activity against Saccharomyces cerevisiae and Candida pseudotropicalis. A bacteriocin was purified from E. durans (isolated from Pandora). The molecular mass of the purified peptide was 6316.89±0.64 Da (Migaw et al., 2013).
Belfiore and colleagues (2010) also isolated four species of Leuconostoc from anchovy (Engraulis anchoita). They suggested that the generated amino acid from fish would be transformed into aroma compounds by Lc. mesenteroides subsp. dextranicum, Lc. carnosum and Lc. mesenteroides subsp. mesenteroides. An anti-Listeria compound was also detected for Leuc. mesenteroides. Two species of LAB; Lb. casei and Lb. plantarum were selected from 84 isolated lactobacilli from the intestine of Persian sturgeon (Acipenser persicus) and Beluga (Huso huso). From the study, both species were able to produce antibacterial substances. They have a high inhibitory potential against Listeria monocytogenes (Ghanbari et al., 2013) (Alonso et al., 2018).
Al Bulushi et al. (2010) conducted a study by storing the white trevally (Pseducaranx dentex), silver seabream (Pagrus auratus) and flathead grey mullet (Mugil cephalus) at 25ᵒC for 15 hours. They mentioned that the highest frequency of LAB was found in the gills of the fish and the lowest in the gut. From that, the frequencies of S. uberis and E. faecium were found the most at 15 hours of storage. Bahmani et al. (2014) studied the effect of delayed icing on the quality of mullet. They investigated the total viable count of LAB and Enterobacteriaceae. From their study, the growth rate of the total viable count population of bacteria decreased at the end of storage. They suggested that the bacteria might be inhibited or killed with the more production of natural preservatives by LAB such as short-chain fatty acid and bacteriocins. This condition may help in maintaining an appropriate pH and protect against pathological changes in fish during storage. Nine LAB were isolated from sardine (Sardina pilchardus) and leatherjacket (Oligoplites saliens). Lb. homohiochii and Lb. farciminis were detected from viscera of sardine and Lb. intestinalis was obtained from viscera of leatherjacket. Further study showed that Lb. homohiochii grew optimally at 40ᵒC, meanwhile Lb. intestinalis grew optimally at 30ᵒC (Poffo et al., 2011). Table 3 shows LAB isolated from invertebrate marine organisms. A study conducted by Iehata et al. (2013) discovered that there were different bacterial communities and bacterial nutritional enzyme activity between both female and male Chilean octopus (Octopus mimus Gould). They suggested the possibility of different feeding habits and the formation of the bacterial community could reflect the environment of the host digestive tract. From this study, Lc. garvieae was found in both male and female octopuses. Eventually, Lc. garvieae and Lc. formosensis isolated from red octopus (Octopus maya) were detected as histamine producing strains. They represented 6% of the total isolates (Gullian et al., 2018). E. faecium, E. hirae, E. faecalis and E. gallinarum were isolated from faeces of black sea urchin (Arbacia lixula), sea urchin (Paracentrotus lividus), purple sea urchin (Sphaerechinus granularis), and sea cucumber (Holothuria mammata and Holothuria sanctori). The investigation showed that E. faecium was obtained as the predominant enterococcal species in the faecal samples. The enterococci showed high resistance to ampicillin, tetracycline and ciprofloxacin (Marinho et al., 2013).

Invertebrate marine organism
A study conducted by Boulares and colleagues (2011) (Wang et al., 2018). Table 4 shows LAB isolated from marine mammals. Firmicutes phylum was composed as the highest in the dugong's faecal content. The bacterial flora in the same group including manatee and dugong may be varied according to several factors such as species, age, habitat, eating habits, digestive tract and tract position. It was unclear whether the seasonal variation can be related to the variation of chemical composition and digestion potential, or changes of the physiological or digestion metabolism of the dugong that related to the ageing of the animal (Tsukinowa et al., 2008). Lactobacillus salivarius isolated from the gastric fluid of bottlenose dolphin (Tursiops truncatus) revealed the potential to suppress the proliferation of enteric pathogen and stimulate tumour necrosis factor production in mammalian myeloid cells. Novel Lactobacillus spp. was isolated and related to Leuconostoc ceti (Diaz et al., 2013).

Marine mammal
Two unidentified cocci were also isolated from the dead seal and porpoise. Comparative 16S rRNA gene sequencing was performed to determine the phylogenetic affinities of the two unknown cocci. Both isolates were 100% identical to each other and from the phylogenetic and phenotypic distinctiveness of the unknown bacterium, it was proposed as Vagococcus fessus (Hoyles et al., 2000).

Marine reptile
Sample from faeces and cloaca were collected from hospitalized sea turtles and the intestine samples were taken from the dead sea turtles. LAB was obtained from the faeces, cloaca and intestine of sea turtles (Caretta caretta). Sample from faeces and cloaca were collected from hospitalized sea turtles and the intestine samples were taken from the dead sea turtles. The location of the sample taken was in Tuscany and Liguria, Italy. From taxonomic composition, it showed that 66% of Firmicutes dominated the faeces sample and 87% from the intestine samples. Vagococcus with 42.3% was the most presented bacteria in the intestine (Abdelrhman et al., 2016). To the best of our knowledge, there were very limited studies conducted on marine reptiles.

Conclusion
Lactic acid bacteria have been isolated from marine animals from various sites of the animal's part in different parts of the geographical area. Various species were isolated from marine animals with the most dominant genus from Lactobacillus. The bacteria inhabit mostly the GIT of the animals. LAB protect the host by producing bacteriocin to control the growth of pathogens. The resistance of LAB towards antibiotics could be useful in playing a role as a probiotic in the medical industry. LAB isolated from the marine environment has the benefits of resilience to temperature and salty environment compared to other LAB strains. However, to the best of our knowledge, there is a lack of study in the discovery of the potential of LAB from marine animals to industries. More studies should be conducted to get the potential LAB to be manipulated into commercial products. Dugong (Dugong dugong) * ni -no further information, Lb. -Lactobacillus, Lc. -Lactococcus Table 4. Lactic acid bacteria isolated from marine mammal