Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

<Emphasis Type="Italic">Lactobacillus futsaii</Emphasis> CS3, a New GABA-Producing Strain Isolated from Thai Fermented Shrimp (<Emphasis Type="Italic">Kung</Emphasis>-<Emphasis Type="Italic">Som</Emphasis>)

Kung-Som is a popular traditional Thai fermented shrimp product. It is rich in glutamic acid, which is the major substrate for the biosynthesis of gamma-aminobutyric acid (GABA) by lactic acid bacteria (LAB). In the present study, LAB from Kung-Som were isolated, screened for GABA formation, and the two isolates that transform glutamic acid most efficiently into GABA were identified. Based on the API-CHL50 fermentation profile and a phylogenetic tree of 16S rDNA sequences, strain CS3 and CS5 were identified as Lactobacillus futsaii, which was for the first time shown to be a promising GABA producer. L. futsaii CS3 was the most efficient microorganism for the conversion of 25 mg/mL monosodium glutamate (MSG) to GABA, with a maximum yield of more than 99% conversion rate within 72 h. The open reading frame (ORF) of the glutamate decarboxylase (gad) gene was identified by PCR. It consists of 1410 bp encoding a polypeptide of 469 amino acids with a predicted molecular weight of 53.64 kDa and an isoelectric point (pI) of 5.56. Moreover, a good quality of the constructed model of L. futsaii CS3 was also estimated. Our results indicate that L. futsaii CS3 could be of interest for the production of GABA-enriched foods by fermentation and for other value-added products.     

Characterization of bacteriocins produced by strains of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> isolated from Minas cheese

Interest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.     

Renewable microbial lipid production from <Emphasis Type="Italic">Oleaginous Yeast</Emphasis>: some surfactants greatly improved lipid production of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Microbial oil is drawing increasing interest worldwide as an alternative non-food oil feedstock for biodiesel industry. Nowadays researchers have been increasingly focused on the improvement of microbial oil production process. Oleaginous yeast Rhodosporidium toruloides (R. toruloides) is considered an important candidate due to its excellent capabilities of lipid accumulation, broad adaptabilities to various carbon substrates, and the potential of co-production of some pigments. In present work, the individual effects of non-ionic, cationic, and anionic surfactant on cell growth and lipid accumulation of R. toruloides were investigated for the first time. Interesting results were noticed when some anionic surfactants were supplemented. The most significant effect was observed with addition of 0.2 % (w/v) sodium lignosulfonate, that biomass concentration, lipid concentration, and lipid yield was increased by 25.1, 44.9, and 15.7 %, respectively. The fatty acid compositions of R. toruloides lipids remained unchanged, which is similar to that of vegetable oils, and is considered potential feedstock for biodiesel preparation.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> as a biocatalyst for biofuel production

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

Functional Properties of <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis> Strains Isolated from Brazilian Goat Milk

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.     

Engineered <Emphasis Type="Italic">E. coli</Emphasis> W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

Background

Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production.

Results

The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l^?1 h^?1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l^?1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l^?1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals.

Conclusion

A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

     

The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H₂O₂. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H₂O₂ exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.     

Survey on proteolytic activity and diversity of proteinase genes in mesophilic lactobacilli

Lactocepins or CEPs are large cell wall bound extracellular proteinases of lactic acid bacteria, involved in protein breakdown and utilization. They are responsible for many health-promoting traits of food products fermented with these organisms, but also essential for probiotic effects of certain strains. Different mesophilic strains selected within the species Lactobacillus zeae, Lb. casei, Lb. rhamnosus, and Lb. plantarum were analyzed for their proteolytic activity towards main fractions of milk proteins—caseins and whey proteins. The strains showing excellent proteolytic features were further examined for presence of corresponding proteinase gene(s). It was found that Lb. zeae LMG17315 possessed catalytic domains of three distinct proteinase genes, unique feature in Lb. casei group, which are similar but not identical to previously characterized prtP and prtR genes. Lb. casei neotype strain ATCC393 was also analysed and based on obtained results its reclassification in taxon Lb. zeae is supported. In addition, we report catalytic domain of prtR-type gene in Lb. plantarum LMG9208, which is first such report in this species, and it is first time that this gene is reported outside Lb. casei group.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Exploitation of genus <Emphasis Type="Italic">Rhodosporidium</Emphasis> for microbial lipid production

Oleaginous microorganisms are receiving significant attention worldwide for their utility in biodiesel production and the potentiality to produce some specialty-type lipids. There is an increasing interest in isolation/adaption of robust microbe strains and design of innovative fermentation processes to make microbial lipid production a more efficient and economically feasible bio-process. Currently, the genus Rhodosporidium has been considered an important candidate, for the reason that several strains belonging to this genus have shown excellent capabilities of lipid accumulation, broad adaptabilities to various substrates, and co-production of some carotenoids. This paper reviews the current trends in the exploitation of Rhodosporidium species for microbial lipid production, including the utilization of various (single or mixed, pure or waste-derived) substrates, progress of genetic modification and metabolic engineering, innovations in fermentation mode, lipid characterizations and their potential applications. Finally, the constraints and perspectives of cultivating Rhodosporidium species for lipid production are also discussed.     

Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.     

An alternative strategy for enhancing lipid accumulation in chlorophycean microalgae for biodiesel production

Oleaginous microalgae are considered as important feedstocks for production of biodiesel. Under nutrient stress conditions, microalgae have the ability to accumulate higher amount of lipids, which can be transesterified for the production of biodiesel. In the present investigation, four different phosphate application strategies were examined in five green microalgae (Tetradesmus obliquus, Tetradesmus lagerheimii, Chlorella vulgaris, Chlorella minutissima, and Chlamydomonas sp.) to achieve higher lipid productivity. Effects of those strategies such as phosphate-sufficient (Control), phosphate-starved approach (PSA), biphasic phosphate-starved approach (BPSA), and sequential phosphate addition (SPA) were studied under batch culture mode. The BPSA emerging as the best in terms of lipid productivity consisted of two biomass harvesting phases, which would lead to an increase in the overall cost of biodiesel production. On the other hand, the SPA with a 1/200th dose of N 11 medium, i.e., 0.4 mg L^?1 of phosphate application in 3-day intervals, also resulted into higher lipid productivity which was equal to BPSA. Fatty acid composition of the biodiesel obtained from the microalgae was analyzed and the fuel characteristics were also evaluated. A profound (~14-fold) reduction in phosphorus requirements under the SPA mode with higher lipid productivity ensured qualitative biodiesel production and a lesser amount of phosphorus release, thus making the process eco-friendly.     

Single cell oil production from a newly isolated <Emphasis Type="Italic">Candida viswanathii</Emphasis> Y-E4 and agro-industrial by-products valorization

Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel and added-value compounds production. To this end, new oleaginous yeast, Candida viswanathii Y-E4 was isolated, characterized and used for single cell oil (SCO) production. Physiologic and nutritional parameters optimization was carried out for improved biomass and lipid production. Y-E4 strain was able to use a wide range of substrates, especially C5 and C6 sugars as well as glycerol and hydrophobic substrates. The fatty acid profile analysis showed that oleic acid was the main component produced using different substrates. Batch and fed-bath fermentation were conducted using glucose as carbon source. Lipid production rate is twice higher in fed-batch culture providing a lipid content of 50 % (w/w). To minimize the SCO production cost, C. viswanathii Y-E4 was evaluated for its capacity to use different agro-industrial by-products for microbial oil production and changes in the fatty acid profile were monitored.     

Production of acrylic acid and propionic acid by constructing a portion of the 3-hydroxypropionate/4-hydroxybutyrate cycle from <Emphasis Type="Italic">Metallosphaera sedula</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Acrylic acid and propionic acid are important chemicals requiring affordable, renewable production solutions. Here, we metabolically engineered Escherichia coli with genes encoding components of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula for conversion of glucose to acrylic and propionic acids. To construct an acrylic acid-producing pathway in E. coli, heterologous expression of malonyl-CoA reductase (MCR), malonate semialdehyde reductase (MSR), 3-hydroxypropionyl-CoA synthetase (3HPCS), and 3-hydroxypropionyl-CoA dehydratase (3HPCD) from M. sedula was accompanied by overexpression of succinyl-CoA synthetase (SCS) from E. coli. The engineered strain produced 13.28 ± 0.12 mg/L of acrylic acid. To construct a propionic acid-producing pathway, the same five genes were expressed, with the addition of M. sedula acryloyl-CoA reductase (ACR). The engineered strain produced 1430 ± 30 mg/L of propionic acid. This approach can be expanded to synthesize many important organic chemicals, creating new opportunities for the production of chemicals by carbon dioxide fixation.     

Optimization of aeration for biodiesel production by <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> grown in municipal wastewater

Despite the significant breakthroughs in research on microalgae as a feedstock for biodiesel, its production cost is still much higher than that of fossil diesel. One possible solution to overcome this problem is to optimize algal growth and lipid production in wastewater. The present study examines the optimization of pretreatment of municipal wastewater and aeration conditions in order to enhance the lipid productivity of Scenedesmus obliquus. Results showed that no significant differences were recorded in lipid productivity of S. obliquus grown in primary settled or sterilized municipal wastewater; however, ultrasound pretreatment of wastewater significantly decreased the lipid production. Whereas, aeration rates of 0.2 vvm significantly increased lipid content by 51 %, with respect to the non-aerated culture, which resulted in maximum lipid productivity (32.5 mg L^?1 day^?1). Furthermore, aeration enrichment by 2 % CO₂ resulted in increase of lipid productivity by 46 % over the CO₂ non-enriched aerated culture. Fatty acid profile showed that optimized aeration significantly enhanced monounsaturated fatty acid production, composed mainly of C18:1, by 1.8 times over the non-aerated S. obliquus culture with insignificant changes in polyunsaturated fatty acid proportion; suggesting better biodiesel characteristics for the optimized culture.     

Human milk and mucosal lacto- and galacto-<Emphasis Type="Italic">N</Emphasis>-biose synthesis by transgalactosylation and their prebiotic potential in <Emphasis Type="Italic">Lactobacillus</Emphasis> species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.     

In vitro assessment of safety and probiotic potential characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from water buffalo mozzarella cheese

The aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).