An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.     

Genetic screening of wine‐related enzymes in Lactobacillus species isolated from South African wines

Aims: The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons. Methods and Results: A total of 120 wine lactobacilli isolates belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus sakei and Lactobacillus paraplantarum were genetically screened for enzyme‐encoding genes using PCR with primers specific for β‐glucosidase, protease, esterase, citrate lyase and phenolic acid decarboxylase. The results of PCR screening showed that the Lactobacillus strains possessed different combinations of enzymes and that some strains did not possess any of the enzymes tested. Confirmation analysis with gene sequencing also showed high similarity of genes with those available in GenBank database. Conclusion: In this study, we have demonstrated the existence of genes coding for wine‐related enzymes in wine lactobacilli that could potentially hydrolyse wine precursors to positively influence wine aroma. Significance and Impact of the Study: An expansion of knowledge on the genetic diversity of wine‐associated lactic acid bacteria will enable the selection of novel malolactic fermentation starter cultures with desired oenological traits for the improvement of the organoleptic quality of the wine, and hence wine aroma.     

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

     

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917^T) and L. pentosus (NCFB 363^T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Identification and probiotic properties of lactobacilli isolated from two different fermented beverages

Purpose

Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals.

Methods

Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential.

Results

Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract.

Conclusion

Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Application of Probiotics in Folate Bio-Fortification of Yoghurt

Folate deficiency is a public health concern affecting all age groups worldwide. The available evidence reveals that adding probiotic bacteria to the yoghurt starter cultures during yoghurt production process under fermentation conditions increases the folate content of yoghurt. The present study was conducted to measure two folate derivatives, i.e., 5-methyltetrahydrofolate and 5-formyltetrahydrofolate, in bio-fortified yoghurt samples including (1) yoghurt containing Streptococcus thermophilus and Lactobacillus bulgaricus, (2) probiotic yoghurt containing Lactobacillus acidophilus LA-5 and Bifidobacterium lactis BB-12, (3) probiotic yoghurt containing native strains of Lactobacillus plantarum 15HN, (4) probiotic yoghurt containing native strains of Lactococcus lactis 44Lac, and (5) probiotic yoghurt containing commercial strains of Lactobacillus plantarum LAT BY PL. During storage at 4 °C for 21 days, the highest levels of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate, which were statistically significant, were detected in the yoghurt made using Lact. plantarum 15HN. Moreover, the highest total folate concentration (1487 ± 96.42 μg/L) was specified in the yoghurt containing Lact. plantarum 15HN on the 7th day. It can be conjectured that this product can be suggested as a proper alternative to synthetic folic acid and may not have the side effects of using synthetic folic acid overdoses.

     

短乳杆菌与植物乳杆菌的发酵特性

【背景】目前对于酸菜发酵的研究主要关注点是植物乳杆菌(Lactobacillus plantarum),有关短乳杆菌(Lactobacillus brevis)在酸菜方面的研究报道很少。【目的】为了挖掘短乳杆菌的发酵性能并开发酸菜发酵剂,将2株短乳杆菌分别与1株植物乳杆菌进行组合并发酵酸菜,分析短乳杆菌对酸菜发酵品质的影响。【方法】分别测定短乳杆菌与植物乳杆菌的单菌株生长产酸性能、耐酸性及亚硝酸盐降解力,并将两菌种组合后发酵酸菜,分析1-7d内酸度、乳酸菌活菌数、亚硝酸盐含量及酸菜质构特性的变化趋势。【结果】相较于短乳杆菌Lb-9-2,短乳杆菌Lb-5-3的生长和产酸速率较慢、酸耐受力较弱,但其亚硝酸盐降解力较强。两株短乳杆菌分别与植物乳杆菌Lp-9-1组合后产酸力显著增强,并在3 d时达到最低pH值(约3.10);植物乳杆菌Lp-9-1的添加使酸菜中总体乳酸菌生长延迟,在5 d时达到最高活菌数;组合菌种的样品中亚硝酸盐含量在1-7 d内变化较为平缓,前5天内两个组合之间差异不显著;接种乳酸菌会降低酸菜硬度和弹性,发酵3d时Lb-5-3/Lp-9-1组合的硬度最大,感官评价得分最高。【...     

Probiotic characteristics of Lactobacillus strains isolated from cheese and their antibacterial properties against gastrointestinal tract pathogens

In the present study, four Lactobacillus strains from the cheese were analyzed for its probiotic potential against enteropathogenic bacteria. The probiotic properties of the selected strains were also analyzed and the selected bacterial strains showed high tolerance in bile salts and organic acid. The strain L. plantarum LP049 showed maximum survival rate (92 ± 4.2% and 93.3 ± 2%) after 3 h of treatment at 0.25% (w/v) bile salts and 0.25% (w/v) organic acid concentrations. The ability of the Lactobacillus strains to adhere to human epithelial cells (HT-29 cell lines) was evaluated and L. plantarum LP049 showed maximum adhesion property (19.2 ± 1.1%) than other tested strains. The Lactobacillus strains produced lactic acid at various concentrations. Compared with other strains, maximum level of lactic acid (3.1 g/L), hydrogen peroxide (4.31 mM) and bacteriocin (31 AU/mg) was detected in LB049. The inhibitory activity of culture supernatant against various bacterial pathogens was observed. The zone of inhibition ranged between 6 ± 2 mm and 23 ± 2 mm. The cell free extract showed activity against, Escherichia coli (ATCC 10536), Salmonella enteritidis (ATCC 13076), Shigella flexneri (ATCC 29903), and Enterococcus faecium (ATCC 8459). Consequently, L. plantarum LP049 may be considered as a potential candidate for the production of novel bioactive metabolites for therapeutic and bio-protective applications.     

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Highly Heterogeneous Probiotic Lactobacillus Species in Healthy Iranians with Low Functional Activities

Background

Lactic acid bacteria (LAB) have been considered as potentially probiotic organisms due to their potential human health properties. This study aimed to evaluate both in vitro and in vivo, the potential probiotic properties of Lactobacillus species isolated from fecal samples of healthy humans in Iran.

Methods and Results

A total of 470 LAB were initially isolated from 53 healthy individual and characterized to species level. Of these, 88 (86%) were Lactobacillus species. Biochemical and genetic fingerprinting with Phene-Plate system (PhP-LB) and RAPD-PCR showed that the isolates were highly diverse consisted of 67(76.1%) and 75 (85.2%) single types (STs) and a diversity indices of 0.994 and 0.997, respectively. These strains were tested for production of adhesion to Caco-2 cells, antibacterial activity, production of B12, anti-proliferative effect and interleukin-8 induction on gut epithelial cell lines and antibiotic resistance against 9 commonly used antibiotics. Strains showing the characteristics consistent with probiotic strains, were further tested for their anti-inflammatory effect in mouse colitis model. Only one L. brevis; one L. rhamnosus and two L. plantarum were shown to have significant probiotic properties. These strains showed shortening the length of colon compared to dextran sulfate sodium and disease activity index (DAI) was also significantly reduced in mouse.

Conclusion

Low number of LAB with potential probiotic activity as well as high diversity of lactobacilli species was evident in Iranian population. It also suggest that specific strains of L. plantarum, L. brevis and L. rhamnosus with anti-inflammatory effect in mouse model of colitis could be used as a potential probiotic candidate in inflammatory bowel disease to decrease the disease activity index.     

Expression of fluorescent proteins in Lactobacillus rhamnosus to study host–microbe and microbe–microbe interactions

Probiotic Lactobacillus strains are widely used to benefit human and animal health, although the exact mechanisms behind their interactions with the host and the microbiota are largely unknown. Fluorescent tagging of live probiotic cells is an important tool to unravel their modes of action. In this study, the implementation of different heterologously expressed fluorescent proteins for the labelling of the model probiotic strains Lactobacillus rhamnosusGG (gastrointestinal) and Lactobacillus rhamnosusGR‐1 (vaginal) was explored. Heterologous expression of mTagBFP2 and mCherry resulted in long‐lasting fluorescence of L. rhamnosusGG and GR‐1 cells, using the nisin‐controlled expression (NICE) system. These novel fluorescent strains were then used to study in vitro aspects of their microbe–microbe and microbe–host interactions. Lactobacillus rhamnosusGG and L. rhamnosusGR‐1 expressing mTagBFP2 and mCherry could be visualized in mixed‐species biofilms, where they inhibited biofilm formation by Salmonella Typhimurium–gfpmut3 expressing the green fluorescent protein. Likewise, fluorescent L. rhamnosusGG and L. rhamnosusGR‐1 were implemented for the visualization of their adhesion patterns to intestinal epithelial cell cultures. The fluorescent L. rhamnosus strains developed in this study can therefore serve as novel tools for the study of probiotic interactions with their environment.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

In vitro antifungal,probiotic and antioxidant properties of novel Lactobacillus plantarum K46 isolated from fermented sesame leaf

This study aimed to describe the diversity of antifungal lactic acid bacteria (LAB) in popular traditional Korean fermented food. A total of 22 LAB strains was selected and subjected to a monophasic identical approach using 16S rRNA gene sequence analysis. Antifungal LAB associated with fermented food was identified as Lactobacillus plantarum (9), Lactobacillus graminis (5), Lactobacillus pentosus (4), Lactobacillus sakei (2), Lactobacillus paraplantarum (1), and Leuconostoc mesenteroides subsp. mesenteroides (1). Novel Lactobacillus plantarum strain K46 exhibited comparatively better antifungal activity against several spoilage fungi, and was deposited in the Korean Collection for Type Cultures (KACC91758P). Antifungal substances from the spent medium in which K46 was cultivated were extracted with ethyl acetate. Antifungal activity was assessed using the broth micro dilution technique. Compounds were characterized based on infrared, ¹³C nuclear magnetic resonance (NMR), and ¹H NMR spectral data. The minimum inhibitory concentration (MIC) of the compounds against Aspergillus clavatus, Aspergillus oryzae, Penicillium chrysogenum and Penicillium roqueforti was 2.5 mg/mL and that against Aspergillus fumigatus, Aspergillus niger, Curvularia lunata and Gibberella moniliformis was 5.0 mg/mL. K46 was able to survive gastrointestinal conditions simulating the stomach and the duodenum passage with the highest percentage of hydrophobicity. In addition, its resistance to hydrogen peroxide and highest hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, with inhibition rates of 43.53 % and 56.88 %, respectively, were to its advantage. An antimicrobial susceptibility pattern was an intrinsic feature of this strain, thus consumption does not represent a health risk to humans. The results showed the potential of K46 strain as an antifungal, probiotic and antioxidant culture, and hence it was determined to be suitable for application in functional foods.     

Antifungal properties of selected lactic acid bacteria and application in the biological control of ochratoxin A producing fungi during cocoa fermentation

Thirteen Lactic acid bacteria strains isolated from fermenting cocoa and seven reference strains were used in order to assess their antifungal properties towards three ochratoxin A (OTA) producing fungi (Aspergillus carbonarius, Aspergillus niger and Aspergillus ochraceus). Furthermore, two of the isolates strains (A19 and A21) identified as belonging to the genus of Pediococcus as well as Lactobacillus plantarum B4496, Lactobacillus brevis 207 and Lactobacillus sanfranciscensis BB12 showed interesting in vitro broad antifungal activities towards the three ochratoxin-producing fungi with inhibition percentages ranging from 15% to 66.7%. Treatment of cell-free supernatant at 100°C affected antifungal activity suggesting that the main compounds responsible for this activity were of proteic nature, and hence could be bacteriocins. Application of isolate A19 in cocoa fermentation as starter inhibited the growth of each of the OTA-producing species. At the end of fermentation in boxes inoculated with A19, A. niger was not detectable while A. carbonarius concentration was found to be 2 Log CFU/g of wet beans. The assessment of the ochratoxin produced during fermentation of cocoa inoculated with A. carbonarius indicated that the use of isolate A19 as starter could reduce their level of growth so as to have only a toxin production of 0.0012 ± 0.0005 μg/kg after 40 days of storage, while this was 2.45 ± 0.35 μg/kg of fermented and dried cocoa beans in the absence of A19. This work is a contribution for the application of biological control of OTA-producing fungi during cocoa production.