Cholesterol homeostasis associated with probiotic supplementation in vivo.

AIMS: To determine the mechanism underlying the serum cholesterol reduction effect by probiotics isolated from local fermented tapioca (Tapai). METHODS AND RESULTS: Lactic acid bacteria strains were isolated and examined for acid tolerance, bile salt resistance and hypocholesterolemic properties. Among the isolates, Lactobacillus plantarum TAR4 showed the highest cholesterol reduction ability (48·01%). The focus in the in vivo trial was to elucidate the cholesterol balance from findings pertaining to serum cholesterol reduction in rat model fed with high fat diet via oral administration. Rats fed with high-cholesterol diet supplemented with Lact. plantarum TAR4 showed significant reduction in serum total cholesterol (29·55%), serum triglyceride (45·31%) and liver triglyceride (23·44%) as compared to high-cholesterol diet (HCD) group. There was a significant increment in faecal triglyceride (45·83%) and faecal total bile acid (384·95%) as compared to HCD group. CONCLUSIONS: The findings showed that probiotic Lact. plantarum TAR4 supplementation reduced the absorption of bile acids for enterohepatic recycling and increased the catabolism of cholesterol to bile acids and not by suppressing the rate of cholesterol synthesis. SIGNIFICANCE AND IMPACT OF STUDY: Probiotic supplements could provide a new nonpharmacological alternative to reduce cardiovascular risk factors.

Current status and application of lactic acid bacteria in animal production systems with a focus on bacteria from honey bee colonies.

Lactic acid bacteria (LAB) are widely distributed in nature and, due to their beneficial effects on the host, are used as probiotics. This review describes the applications of LAB in animal production systems such as beekeeping, poultry, swine and bovine production, particularly as probiotics used to improve health, enhance growth and reproductive performance. Given the importance of honeybees in nature and the beekeeping industry as a producer of healthy food worldwide, the focus of this review is on the coexistence of LAB with honeybees, their food and environment. The main LAB species isolated from the beehive and their potential technological use are described. Evidence is provided that 43 LAB bacteria species have been isolated from beehives, of which 20 showed inhibition against 28 species of human and animal pathogens, some of which are resistant to antibiotics. Additionally, the presence of LAB in the beehive and their relationship with antibacterial properties of honey and pollen is discussed. Finally, we describe the use of lactic bacteria from bee colonies and their antimicrobial effect against foodborne pathogens and human health. This review broadens knowledge by highlighting the importance of honeybee colonies as suppliers of LAB and functional food.

Production of γ-aminobutyric acid (GABA) by lactic acid bacteria strains isolated from traditional, starter-free dairy products made of raw milk.

γ-Aminobutyric acid (GABA), an amino acid not used in protein synthesis, intervenes in several physiological functions and has both diuretic and calming effects in humans. Lactic acid bacteria (LAB) strains that produce GABA could be exploited for the manufacture of health-promoting GABA-enriched dairy products. In this study, 262 LAB strains isolated from traditional dairy products made from raw milk without starter cultures were screened for GABA production in culture media supplemented with 1% monosodium glutamate (MSG) using an enzymatic (GABase) method. About half of the strains (123) were found to be GABA producers. Of these, 24, among which were 16 Lactococcus lactis subsp. lactis and three Streptococcus thermophilus strains, produced >1 mM of GABA (range 1.01-2.81 mM) and were selected for further characterisation. GABA production was confirmed in most strains by culturing in 5 mM MSG followed by HPLC quantification. A majority of the strains were confirmed to be GABA producers by this method, although lower production levels were recorded. Using species-specific primers, the gene encoding glutamate decarboxylase (GAD) was PCR-amplified in all but one of the GABA producers analysed. Amplicons sequences were compared to one another and to those held in databases. Except for one Lactobacillus brevis strain, none of the 24 GABA producers investigated produced toxic biogenic amines, such as tyramine, histamine or cadaverine. They were therefore considered safe. Either alone, in mixtures, or in combination with industrial starter or adjunct cultures, these strains might be useful in the development of health-oriented dairy products.

Novel solid-state fermentation of bee-collected pollen emulating the natural fermentation process of bee bread.

Structure of lactic acid bacteria biota in ivy flowers, fresh bee-collected pollen (BCP), hive-stored bee bread, and honeybee gastrointestinal tract was investigated. Although a large microbial diversity characterized flowers and fresh BCP, most of lactic acid bacteria species disappeared throughout the bee bread maturation, giving way to Lactobacillus kunkeei and Fructobacillus fructosus to dominate long stored bee bread and honeybee crop. Adaptation of lactic acid bacteria was mainly related to species-specific, and, more in deep, to strain-specific features. Bee bread preservation seemed related to bacteria metabolites, produced especially by some L. kunkeei strains, which likely gave to lactic acid bacteria the capacity to outcompete other microbial groups. A protocol to ferment BCP was successfully set up, which included the mixed inoculum of selected L. kunkeei strains and Hanseniaspora uvarum AN8Y27B, almost emulating the spontaneous fermentation of bee bread. The strict relationship between lactic acid bacteria and yeasts during bee bread maturation was highlighted. The use of the selected starters increased the digestibility and bioavailability of nutrients and bioactive compounds naturally occurring in BCP. Our biotechnological protocol ensured a product microbiologically stable and safe. Conversely, raw BCP was more exposed to the uncontrolled growth of yeasts, moulds, and other bacterial groups.

Control of Autochthonous Spoilage Lactic Acid Bacteria in Apple and Orange Juices by Sensorially Accepted Doses of Citrus Spp. Essential Oils Combined with Mild Heat Treatments.

This study assessed the compromised acceptance threshold (CAT) and rejection threshold (RT) of Citrus lemon (CLEO) and Citrus reticulata essential oil (CREO) in apple and orange juices. The efficacy of CLEO and CREO concentrations below the RT were evaluated alone and combined with mild heat treatment (MHT) (54 °C, up to 12 min) to inactivate the autochthonous spoilage bacteria Lactobacillus brevis, Lactobacillus plantarum, and Leuconostoc mesenteroides in apple and orange juices. The CAT of CLEO and CREO varied from 0.15 to 0.17 µL/mL in orange and apple juices. The RT of CLEO was approximately 0.58 µL/mL in apple and orange juices, and the RT of CREO was 0.68 µL/mL in both juices. When CLEO and CREO were assayed alone, the highest concentration (0.50 µL/mL) decreased counts of all strains approximately 2 log10 CFU/mL after 12 min of exposure to 54 °C. All concentrations of CLEO or CREO in combination with MHT acted synergistically against L. brevis, L. plantarum, and L. mesenteroides. Decreases in counts varied with the strain, CLEO and CREO concentrations, juice type, and exposure time to the combined treatment. CREO was more effective than CLEO in combination with MHT against the strains in apple and orange juices. Effective combinations of CLEO or CREO with MHT to control the autochthonous spoilage bacteria did not compromise the quality parameters (°Brix, pH, and titratable acidity) that characterize unsweetened juices. These results indicate CLEO or CREO at concentrations below the sensory RT in combination with MHT as a feasible technology to control autochthonous spoilage bacteria in fresh fruit juices. PRACTICAL APPLICATION: The present study provides novel information concerning the efficacy of sensorially accepted doses of CLEO and CREO combined with MHT against autochthonous spoilers in fruit juice. The valuable synergistic effects that can be observed when combining CLEO and CREO with MHT reveal a feasible preservation technology and alternative to traditional treatments that are successful because they help reduce treatment intensity, thereby avoiding adverse effects on the sensory, physicochemical, and nutritional properties of these products.

Characterization and Antibacterial Activity Against Helicobacter pylori of Lactic Acid Bacteria Isolated from Thai Fermented Rice Noodle.

A total of 32 lactic acid bacteria (LAB) were isolated from Khanom-jeen, a Thai traditional fermented rice noodle. They belonged to the genus Leuconostoc (Ln), Lactobacillus (Lb), Enterococcus (E), Lactococcus (Lc), and Weissella (W), based on their phenotypic characteristics and 16S rRNA gene sequence analyses. The strains were identified as Ln. pseudomesenteroides (group 1, two strains), Ln. citreum (group 2, three strains), Ln. lactis (group 3, three strains), Lb. paracasei subsp. tolerans (group 4, two strains), E. faecium (group 5, three strains), Lc. lactis subsp. lactis (group 6, one strain), W. confusa (group 7, six strains), Lb. fermentum (group 8, seven strains), and Lb. plantarum subsp. plantarum and Lb. pentosus (group 9, five strains). Fifteen strains exhibited the inhibitory activity against Helicobacter pylori clinical isolates by spot-on-lawn method. Lb. fermentum P43-01 resisted to bile acids showed the broad spectrum of antimicrobial activity against H. pylori strains MS83 and BK364. These antagonistic effects were associated with proteinaceous compounds which are sensitive to α-chymotrypsin and pepsin. Results indicated that production of bacteriocin-like substances of selected strain might be the significant mechanism that exerted the inhibition on H. pylori. A potential strain could be used as probiotics in alternative or adjunctive therapy for a patient suffering from H. pylori infection.

Physicochemical responses and microbiological changes of bream (Megalobrama ambycephala) to pectin based coatings enriched with clove essential oil during refrigeration.

The effectiveness of pectin coatings enriched with clove essential oil (CEO), as new edible coatings were investigated to preserve bream (Megalobrama ambycephala) fillets during refrigeration over a period of 15 days. All samples were analyzed for physicochemical (pH, PV, TBA and TVB-N), microbiological (Total viable count, Psychrophilic bacteria, Lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp., H2S producing bacteria) and organoleptic attributes. The results revealed that the CEO incorporation reduced the extent of lipid oxidation, as judged by PV, TBA and TVB-N, thus extending the shelf life of bream fillets by at least 15 days. Moreover, the application of pectin coatings with CEO improved the weight loss, water holding capacity, textural and color attributes of the bream samples significantly compared to untreated sample. Pectin coating along with CEO was effective in inhibiting bacterial growth especially in gram-negative bacteria, while the growth of lactic acid bacteria remained constant for most of the storage period. The effect on the microorganisms during storage was in accordance with biochemical indexes of the quality, representing the viability of these coatings for bream preservation. Thus, the coatings developed in present study could inhibit the development of lipid oxidation during cold storage, representing an option as a seafood preservative.

Genetic diversity and antimicrobial activity of lactic acid bacteria in the preparation of traditional fermented potato product 'tunta'.

Fermentation microorganisms, lactic acid bacteria (LAB) and yeast from 12 samples of tunta production chain were quantified, from the native potatoes used by the process fermentation of potatoes in the river up to the final product. During fermentation, the LAB population steadily increased from 3 to 4 to 8 log CFU/g during the first 8 days in the river and the yeast population increased from 2 to 3 to 3-4 log CFU/g. Overall, 115 LAB strains were isolated using a culture-dependent method. Molecular techniques and 16S rRNA gene sequencing enabled the identification of native species. In LAB isolates, members of the Lactobacillaceae (64%), Leuconostocaceae (9%) and Enterococcaceae (2%) families were identified. The most prevalent LAB species in the tunta production chain was Lactobacillus curvatus, followed by Leuconostoc mesenteroides and Lactobacillus sakei, Lactobacillus brevis and Enterococcus mundtii were also present. Only 13 LAB strains showed anti-listerial activity, and one of them, identified as En. mundtii DSM 4838T [MG031213], produced antimicrobial compounds that were determined to be proteins after treatment with proteolytic enzymes. Based on these results, we suggest that traditional fermented product-derived LAB strains from specific environments could be selected and used for technological application to control pathogenic bacteria and naturally protect food from post-harvest deleterious microbiota.

In Vitro Evaluation of the Probiotic and Safety Properties of Bacteriocinogenic and Non-Bacteriocinogenic Lactic Acid Bacteria from the Intestines of Nile Tilapia and Common Carp for Their Use as Probiotics in Aquaculture.

In this study, seven bacteriocinogenic and non-bacteriocinogenic LAB strains previously isolated from the intestines of Nile tilapia and common carp and that showed potent antibacterial activity against host-derived and non-host-derived fish pathogens were assayed for their probiotic and safety properties so as to select promising candidates for in vivo application as probiotic in aquaculture. All the strains were investigated for acid and bile tolerances, transit tolerance in simulated gastrointestinal conditions, for cell surface characteristics including hydrophobicity, co-aggregation and auto-aggregation, and for bile salt hydrolase activity. Moreover, haemolytic, gelatinase and biogenic amine-producing abilities were investigated for safety assessment. The strains were found to be tolerant at low pH (two strains at pH 2.0 and all the strains at pH 3.0). All of them could also survive in the presence of bile salts (0.3% oxgall) and in simulated gastric and intestinal juices conditions. Besides, three of them were found to harbour the gtf gene involved in pH and bile salt survival. The strains also showed remarkable cell surface characteristics, and 57.14% exhibited the ability to deconjugate bile salts. When assayed for their safety properties, the strains prove to be free from haemolytic activity, gelatinase activity and they could neither produce biogenic amines nor harbour the hdc gene. They did not also show antibiotic resistance, thus confirming to be safe for application as probiotics. Among them, Lactobacillus brevis 1BT and Lactobacillus plantarum 1KMT exhibited the best probiotic potentials, making them the most promising candidates.

Effects of levan-type fructan on growth performance, nutrient digestibility, diarrhoea scores, faecal shedding of total lactic acid bacteria and coliform bacteria, and faecal gas emission in weaning pigs.

BACKGROUND: The use of antibiotics as growth promoters in feed has been fully or partially banned in several countries. The objective of this study was to evaluate effects of levan-type fructan on growth performance, nutrient digestibility, faecal shedding of lactic acid bacteria and coliform bacteria, diarrhoea scores, and faecal gas emission in weaning pigs. A total of 144 weaning pigs [(Yorkshire × Landrace) × Duroc] were randomly allocated to four diets: corn-soybean meal-based diets supplemented with 0, 0.1, 0.5, or 1.0 g kg-1 levan-type fructan during this 42-day experiment. RESULTS: During days 0 to 21 and 0 to 42, average daily gain and average daily feed intake were linearly increased (P < 0.01) with increasing dietary levan-type fructan inclusion. The apparent total tract digestibility of dry matter, crude protein, and gross energy were linearly increased (P < 0.001) with increasing dietary levan-type fructan content. With increasing levels of levan-type fructan, faecal lactic acid bacteria counts were linearly increased (P = 0.001). CONCLUSION: The results indicate that dietary supplementation with increasing levan-type fructan enhanced growth performance, improved nutrient digestibility, and increased faecal lactic acid bacteria counts in weaning pigs linearly. © 2017 Society of Chemical Industry.

Isolation and molecular identification of lactic acid bacteria from King grass and their application to improve the fermentation quality of sweet Sorghum.

The aim of the present study was isolation and molecular identification of lactic acid bacteria from King grass and their application to improve the fermentation quality of sweet Sorghum. Seventy-six strains of LAB were isolated; five strains were selected for Physiological and morphological tests and 16S rRNA sequencing. All five strains grew at different pH 3.5-8.0, different temperature 35, 40, 45, 50 °C and different NaCl concentrations 3, 6.5, 9.5%. Strains HDASK were identified Lactobacillus plantarum and SK3907, SK2A32, SK3A42 and ASKDD Pediococcus acidilactici. Three isolated strains and one commercial strain were added to sweet sorghum. Silage was prepared of four treatments and one control with three replicates as control (SKC, adding 2 ml/kg sterilizing water), L. plantarum commercial bacteria (SKP), L. plantarum (HDASK) isolated from King grass (SKA), P. acidilactici (SK3907) isolated from King grass (SKB) and P. acidilactici (ASKDD) isolated from King grass (SKD). All silage were prepared using polyethylene terephthalate bottles, and incubated at room temperature for different ensiling days. The level of pH, acetic acid, NH3-N, water soluble carbohydrate and butyric acid was significantly (P < 0.05) decreased. Lactic acid, ethanol and propionic acid (PA) was significantly (P < 0.05) increased in treatments compared to control. The dry matter, propionic acid neutral detergent fiber, acid detergent fiber did not significantly (P < 0.05) differ among the treatments but the values were increased and decreased. The number of yeast, mold and LAB were significantly (P < 0.05). It is suggested that the supplementation of LAB could enhanced the fermentation quality of sweet Sorghum silage.

Diversity of lactic acid bacteria from Miang, a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract.

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.

Diversity of lactic acid bacteria associated with fresh coffee cherries in Taiwan.

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.

Honeybees and beehives are rich sources for fructophilic lactic acid bacteria.

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics.