Multistimuli responsive microcapsules produced by the prilling/vibration technique for targeted colonic delivery of probiotics.

This study aimed to microencapsulate the probiotic strain Lactiplantibacillus plantarum 4S6R (basonym Lactobacillus plantarum) in both microcapsules and microspheres by prilling/vibration technique. A specific polymeric mixture, selected for its responsiveness to parallel colonic stimuli, was individuated as a carrier of microparticles. Although the microspheres were consistent with some critical quality parameters, they showed a low encapsulation efficiency and were discarded. The microcapsules produced demonstrated high yields (97.52%) and encapsulation efficiencies (90.06%), with dimensional analysis and SEM studies confirming the desired size morphology and structure. The results of thermal stress tests indicate the ability of the microcapsules to protect the probiotic. Stability studies showed a significant advantage of the microcapsules over non-encapsulated probiotics, with greater stability over time. The release study under simulated gastrointestinal conditions demonstrated the ability of the microcapsules to protect the probiotics from gastric acid and bile salts, ensuring their viability. Examination in a simulated faecal medium revealed the ability of the microcapsules to release the bacteria into the colon, enhancing their beneficial impact on gut health. This research suggests that the selected mixture of reactive polymers holds promise for improving the survival and efficacy of probiotics in the gastrointestinal tract, paving the way for the development of advanced probiotic products.

Selection of Gut-Resistant Bacteria and Construction of Microbial Consortia for Improving Gluten Digestion under Simulated Gastrointestinal Conditions.

This work aimed to define the microbial consortia that are able to digest gluten into non-toxic and non-immunogenic peptides in the human gastrointestinal tract. METHODS: 131 out of 504 tested Bacillus and lactic acid bacteria, specifically Bacillus (64), lactobacilli (63), Pediococcus (1), and Weissella (3), showed strong gastrointestinal resistance and were selected for their PepN, PepI, PepX, PepO, and PepP activities toward synthetic substrates. Based on multivariate analysis, 24 strains were clearly distinct from the other tested strains based on having the highest enzymatic activities. As estimated by RP-HPLC and nano-ESI-MS/MS, 6 cytoplasmic extracts out of 24 selected strains showed the ability to hydrolyze immunogenic epitopes, specifically 57-68 of α9-gliadin, 62-75 of A-gliadin, 134-153 of γ-gliadin, and 57-89 (33-mer) of α2-gliadin. Live and lysed cells of selected strains were combined into different microbial consortia for hydrolyzing gluten under gastrointestinal conditions. Commercial proteolytic enzymes (Aspergillusoryzae E1, Aspergillusniger E2, Bacillussubtilis Veron HPP, and Veron PS proteases) were also added to each microbial consortium. Consortium activity was evaluated by ELISA tests, RP-HPLC-nano-ESI-MS/MS, and duodenal explants from celiac disease patients. RESULTS: two microbial consortia (Consortium 4: Lactiplantibacillus (Lp.) plantarum DSM33363 and DSM33364, Lacticaseibacillus (Lc.) paracasei DSM33373, Bacillussubtilis DSM33298, and Bacilluspumilus DSM33301; and Consortium 16: Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, Limosilactobacillusreuteri DSM33374, Bacillusmegaterium DSM33300, B.pumilus DSM33297 and DSM33355), containing commercial enzymes, were able to hydrolyze gluten to non-toxic and non-immunogenic peptides under gastrointestinal conditions. CONCLUSIONS: the results of this study provide evidence that selected microbial consortia could potentially improve the digestion of gluten in gluten-sensitive patients by hydrolyzing the immunogenic peptides during gastrointestinal digestion.

Prototype Gluten-Free Breads from Processed Durum Wheat: Use of Monovarietal Flours and Implications for Gluten Detoxification Strategies.

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins' primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.

Diet influences the functions of the human intestinal microbiome.

Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in 27 fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three interleukins (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.

Wholemeal wheat flours drive the microbiome and functional features of wheat sourdoughs.

This study aimed to evaluate the effect of soft (Triticum aestivum) and durum (Triticum durum) wheat flours at different extraction rate (type 00, 0, 1 and 2) and wholemeal flours on the microbial composition and functionality of type I sourdough. Enterobacteriaceae constituted the main component of the microbiome of refined soft and durum wheat flours. On the contrary, wholemeal durum wheat flour harboured mainly Xanthomonadaceae. Differences were also found between the soft and durum wheat flours. After 8 h of fermentation (1 day), a different behaviour of the microbiome components was observed. All the mature sourdoughs harboured a core microbiome constituted by 4 species (Pediococcus pentosaceus, Lactobacillus brantae, Pediococcus argentinicus and Weissella cibaria). Based on the type of flour, the relative abundance of each core species differed among sourdoughs. In addition, other dominant lactic acid bacteria species were variously detected in sourdoughs. Mature sourdoughs showed marked variations for the concentrations of glucose, fructose, maltose, lactic and acetic acids, ethanol and free amino acids (FAA). Specific correlations (r > 0.7; FDR < 0.05) were found between the microbiome and total phenols, fibres and metabolome of mature sourdoughs. Breads made by using wholemeal soft wheat or durum wheat doughs started by wholemeal wheat flour-based sourdoughs were characterized by the highest total amount of free cations (Ca2+, Zn2+, Fe2+, Mg2+), antioxidant activity and improved sensory traits.

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200.

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up- or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.

Salivary microbiota and metabolome associated with celiac disease.

This study aimed to investigate the salivary microbiota and metabolome of 13 children with celiac disease (CD) under a gluten-free diet (treated celiac disease [T-CD]). The same number of healthy children (HC) was used as controls. The salivary microbiota was analyzed by an integrated approach using culture-dependent and -independent methods. Metabolome analysis was carried out by gas chromatography-mass spectrometry-solid-phase microextraction. Compared to HC, the number of some cultivable bacterial groups (e.g., total anaerobes) significantly (P < 0.05) differed in the saliva samples of the T-CD children. As shown by community-level catabolic profiles, the highest Shannon's diversity and substrate richness were found in HC. Pyrosequencing data showed the highest richness estimator and diversity index values for HC. Levels of Lachnospiraceae, Gemellaceae, and Streptococcus sanguinis were highest for the T-CD children. Streptococcus thermophilus levels were markedly decreased in T-CD children. The saliva of T-CD children showed the largest amount of Bacteroidetes (e.g., Porphyromonas sp., Porphyromonas endodontalis, and Prevotella nanceiensis), together with the smallest amount of Actinobacteria. T-CD children were also characterized by decreased levels of some Actinomyces species, Atopobium species, and Corynebacterium durum. Rothia mucilaginosa was the only Actinobacteria species found at the highest level in T-CD children. As shown by multivariate statistical analyses, the levels of organic volatile compounds markedly differentiated T-CD children. Some compounds (e.g., ethyl-acetate, nonanal, and 2-hexanone) were found to be associated with T-CD children. Correlations (false discovery rate [FDR], <0.05) were found between the relative abundances of bacteria and some volatile organic compounds (VOCs). The findings of this study indicated that CD is associated with oral dysbiosis that could affect the oral metabolome.

Fermentation and proteome profiles of Lactobacillus plantarum strains during growth under food-like conditions.

This study aimed at investigating the proteomic adaptation of Lactobacillus plantarum strains. Cultivation of L. plantarum strains under food-like conditions (wheat flour hydrolyzed, whey milk, tomato juice) affected some metabolic traits (e.g., consumption of carbohydrates and synthesis of organic acids) compared to de Man, Rogosa and Sharpe (MRS) broth. The analysis of the fermentation profile showed that the highest number of carbon sources metabolized by L. plantarum strains was found using cells cultivated in media containing low concentration of glucose or no glucose at all. The proteomic maps of the strains were comparatively determined after growth on MRS broth and under food-like conditions. The amount of proteins depended on strain and, especially, on culture conditions. Proteins showing decreased or increased amounts under food-like conditions were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Changes of the proteome concerned proteins that are involved in carbohydrate transport and metabolism, energy metabolism, Sec-dependent secretion system, stress response, nucleotide metabolism, regulation of nitrogen metabolism, and protein biosynthesis. A catabolic repression by glucose on carbohydrate transport and metabolism was also found. The characterization of the proteomes in response to changing environmental conditions could be useful to get L. plantarum strains adapted for specific applications. BIOLOGICAL SIGNIFICANCE: Microbial cell performance during food biotechnological processes has become one of the greatest concerns all over the world. L. plantarum is a lactic acid bacterium with a large industrial application for fermented foods or functional foods (e.g., probiotics). The present study compared the fermentation and proteomic profiling of L. plantarum strains during growth under food-like conditions and under optimal laboratory conditions (MRS broth). This study provides specific mechanisms of proteomic adaptation involved in the microbial performances (carbohydrates utilization, energy metabolism, stress resistance, etc.) affecting the main biotechnological tracts of L. plantarum strains. The finding of this study provides evidences that may be exploited to get strains adapted for specific applications in food biotechnology.

Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified.

This study aimed at investigating the fecal microbiota and metabolome of children with Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and autism (AD) in comparison to healthy children (HC). Bacterial tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) of the 16S rDNA and 16S rRNA analyses were carried out to determine total bacteria (16S rDNA) and metabolically active bacteria (16S rRNA), respectively. The main bacterial phyla (Firmicutes, Bacteroidetes, Fusobacteria and Verrucomicrobia) significantly (P<0.05) changed among the three groups of children. As estimated by rarefaction, Chao and Shannon diversity index, the highest microbial diversity was found in AD children. Based on 16S-rRNA and culture-dependent data, Faecalibacterium and Ruminococcus were present at the highest level in fecal samples of PDD-NOS and HC children. Caloramator, Sarcina and Clostridium genera were the highest in AD children. Compared to HC, the composition of Lachnospiraceae family also differed in PDD-NOS and, especially, AD children. Except for Eubacterium siraeum, the lowest level of Eubacteriaceae was found on fecal samples of AD children. The level of Bacteroidetes genera and some Alistipes and Akkermansia species were almost the highest in PDD-NOS or AD children as well as almost all the identified Sutterellaceae and Enterobacteriaceae were the highest in AD. Compared to HC children, Bifidobacterium species decreased in AD. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of free amino acids and volatile organic compounds of fecal samples were markedly affected in PDD-NOS and, especially, AD children. If the gut microbiota differences among AD and PDD-NOS and HC children are one of the concomitant causes or the consequence of autism, they may have implications regarding specific diagnostic test, and/or for treatment and prevention.

Effect of lactose on gut microbiota and metabolome of infants with cow's milk allergy.

Allergic infants have an unusual gastrointestinal microbiota with low numbers of Bifidobacterium/Lactobacilli and high levels of Clostridium, staphylococci and Escherichia coli. Hydrolyzed formula used to treat these infants is deprived of lactose that instead may influence the gut microbial composition. The aim of the present study is to investigate the influence of lactose on the composition of the gut microbiota and metabolome of infants with cow's milk allergy. Infants prospectively enrolled received an extensively hydrolyzed formula with no lactose for 2 months followed by an identical lactose-containing formula for an additional 2 months. Healthy, age-gender-matched infants were used as controls. The following determinations were performed before and after the introduction of lactose in the diet: enumeration of cells present in the feces using FISH, counts of viable bacterial cells and gas-chromatography mass spectrometry/solid-phase microextraction analysis. The addition of lactose to the diet significantly increases the counts of Bifidobacteria and lactic acid bacteria (p < 0.01), decreases that of Bacteroides/clostridia (p < 0.05) reaching counts found in healthy controls; lactose significantly increases the concentration of total short-chain fatty acids (p < 0.05). The addition of lactose to an extensively hydrolyzed formula is able to positively modulate the composition of gut microbiota by increasing the total fecal counts of Lactobacillus/Bifidobacteria and decreasing that of Bacteroides/Clostridia. The positive effect is completed by the increase of median concentration of short chain fatty acids, especially for acetic and butyric acids demonstrated by the metabolomic analysis.

Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I.

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillus plantarum. Seven strains were singly used as sourdough type I starters under daily back-slopping propagation (ten days) using wheat flour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of 9.13-9.46 log cfu g(-1)) between and within started sourdoughs, as well as the acidifying activity (median values of 1.24-1.33). After three days also the control sourdough (unstarted) had the same values. As shown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintained elevated cell numbers (ca. 9 log cfu g(-1)) throughout ten days. The other two strains progressively decreased to less than 5 log cfu g(-1). As identified by partial sequencing of 16S rRNA and recA genes, L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated the flour microbiota. Monitoring of lactic acid bacteria during sourdough propagation was carried out by culture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except for the sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain of L. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiae and Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1. The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughs containing persistent starters behaved similarly and their profiles were clearly differentiated from the others. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previously shown to be persistent (Siragusa et al., 2009), were used as the mixed starter to produce a wheat flour sourdough. Both strains cohabited and dominated during ten days of propagation.

Fermented goats' milk produced with selected multiple starters as a potentially functional food.

A screening among five lactic acid bacteria, used alone or in combination, led to select a mixed starter (Streptococcus thermophilus CR12, Lactobacillus casei LC01, Lactobacillus helveticus PR4, Lactobacillus plantarum 1288) capable to produce a fermented goats' milk containing gamma-aminobutyric acid (GABA) and angiotensin-I converting enzyme (ACE)-inhibitory peptides. The fermented milk was characterized by cell counts of lactic acid bacteria not lower than 7.0 log cfu g(-1), even after 45 days of storage at 4 degrees C. Fermentation of goats' milk resulted in the production of ca. 28 mg kg(-1) of GABA. Furthermore the fermented goats' milk had an in vitro ACE-inhibitory activity of ca. 73%. Prolonged cold storage did not significantly affect both the concentration of GABA and the ACE-inhibitory activity. Moreover, the taurine content did not significantly vary during both fermentation and the entire storage period.

Taxonomic structure and monitoring of the dominant population of lactic acid bacteria during wheat flour sourdough type I propagation using Lactobacillus sanfranciscensis starters.

The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g(-1). Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.

Selection and use of autochthonous mixed starter for lactic acid fermentation of carrots, French beans or marrows.

Strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella soli/Weissella koreensis, Enterococcus faecalis, Pediococcus pentosaceus and Lactobacillus fermentum were identified from raw carrots, French beans and marrows by partial 16S rRNA gene sequence. L. plantarum M1, Leuc. mesenteroides C1 and P. pentosaceus F4 were selected based on the rates of growth and acidification in vegetable juice media, and used as the autochthonous mixed starter for the fermentation of carrots, French beans or marrows. An allochthonous starter, consisting of the same species, was also used for fermentation. A two-step fermentation process (1 day at 25 degrees C and 7 days at 15 degrees C) in brine (1% w/v) followed by storage at room temperature in olive oil until 40 days was set up. Unstarted vegetables subjected to the same treatments were used as the controls. Cell numbers of lactic acid bacteria in the started vegetables were ca. 10,000 (autochthonous starter) and 1000 (allochthonous starter) times higher than unstarted samples throughout the process. When fermented with the autochthonous starter, carrots, French beans or marrows were characterized by the rapid decrease of pH (<4.5), marked consumption of fermentable carbohydrates, and inhibition of Enterobacteriaceae and yeasts. Fermentation with the allochthonous starter did not acidify and inhibit bacteria and yeasts so rapidly. After 40 days, carrots, French beans and marrows fermented with the autochthonous starter had significantly (P<0.05) higher total concentration of vitamin C (ascorbate+dehydroascorbate) with respect to those fermented with the allochthonous starter and, especially unstarted vegetables. The same was found for the indexes of color. Firmness of both started vegetables was higher than unstarted vegetables. Sensory analysis differentiated started vegetables. Carrots and French beans fermented with the autochthonous starter were, especially, appreciated for fragrance. Appearance was the sensory attribute that mainly distinguished marrows fermented with the autochthonous starter.

Survival and persistence of Lactobacillus plantarum 4.1 and Lactobacillus reuteri 3S7 in the gastrointestinal tract of pigs.

Lactobacillus sp. are important inhabitants of the intestines of animals. They are also largely used as probiotics for both humans and animals. To exert beneficial effects, lactobacilli have to survive through the gastrointestinal transit. Based on bile-salt resistance, pH tolerance, antimicrobial activity and heat resistance, Lactobacillus plantarum 4.1 and Lactobacillus reuteri 3S7 were previously selected and used as probiotic additives in pelleted feeding trials. Both strains were fed to pigs (sows and piglets) at a cell number of ca. 10(10) viable cells per day. A polyphasic approach, comprising growth on selective media, Biolog System analysis, 16S rRNA gene sequencing and RAPD-PCR typing, was used to identify and differentiate L. plantarum 4.1 and L. reuteri 3S7 from other faecal Lactobacillus sp., L. plantarum 4.1 and L. reuteri 3S7 had the capacity to survive during the gastrointestinal transit and were found in the feaces at a cell number of 6-8 log cfu/g. Their persistence was shown after 6 days from the administration. Compared to untreated pigs, the administration of L. plantarum 4.1 and L. reuteri 3S7 significantly (P<0.05) decreased the population of Enterobacteriaceae. Besides, the beta-glucuronidase activity of all pigs decreased of ca. 23.0% during administration. The findings of this study showed that L. plantarum 4.1 and L. reuteri 3S7 have the potential to be used as probiotic additives in pelleted feed for pigs.

Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding.

Thirty-five isolates from pig feces were identified as Lactobacillus reuteri (12 strains), Lactobacillus mucosae (7), Lactobacillus plantarum (6), Lactobacillus kitasatonis (3), Lactobacillus rossiae (2), Lactobacillus ultunensis (2), Lactobacillus crispatus (2), and Lactobacillus intestinalis (1) by partial sequence analysis of the 16S rRNA. All isolates were detected at 8-9 log CFU g(-1). Preliminarily, strains were selected based on resistance to heat treatments (ca. 70 degrees C for 10 s). The decrease in viability for some L. reuteri, L. mucosae, L. plantarum, L. kitasatonis, and L. rossiae strains was lower than 1 log cycle. Selected strains were further characterized for acid and bile salt resistance, and antibacterial activity. Except for L. kitasatonis, tolerance to simulated gastric and intestinal conditions was enhanced for all strains by addition of reconstituted skimmed milk. Antibacterial activity was found against Gram-positive and -negative potential pathogens. L. reuteri 8.1, 3S7, 6.2, and 1.2, L. mucosae 1.1R, L. plantarum 4.1, and L. rossiae 4.4 were freeze-dried and mixed (1%, w/w) into pig feed before pelleting. After pelleting, pig feed contained 10-9 log CFU kg(-1) of lactobacilli. L. plantarum 4.1, and L. reuteri 3S7 were selected based on their bile salt resistance, pH tolerance, antimicrobial activity and heat resistance. The findings in this study provide a strong basis for exploring the potential of porcine lactobacilli isolates to be used in pelleted feeding as probiotic additives.