High-Yield Preparation of American Oyster Defensin (AOD) via a Small and Acidic Fusion Tag and Its Functional Characterization.

The marine peptide, American oyster defensin (AOD), is derived from Crassostrea virginica and exhibits a potent bactericidal effect. However, recombinant preparation has not been achieved due to the high charge and hydrophobicity. Although the traditional fusion tags such as Trx and SUMO shield the effects of target peptides on the host, their large molecular weight (12-20 kDa) leads to the yields lower than 20% of the fusion protein. In this study, a short and acidic fusion tag was employed with a compact structure of only 1 kDa. Following 72 h of induction in a 5 L fermenter, the supernatant exhibited a total protein concentration of 587 mg/L. The recombinant AOD was subsequently purified through affinity chromatography and enterokinase cleavage, resulting in the final yield of 216 mg/L and a purity exceeding 93%. The minimum inhibitory concentrations (MICs) of AOD against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus galactis ranged from 4 to 8 µg/mL. Moreover, time-killing curves indicated that AOD achieved a bactericidal rate of 99.9% against the clinical strain S. epidermidis G-81 within 0.5 h at concentrations of 2× and 4× MIC. Additionally, the activity of AOD was unchanged after treatment with artificial gastric fluid and intestinal fluid for 4 h. Biocompatibility testing demonstrated that AOD, at a concentration of 128 µg/mL, exhibited a hemolysis rate of less than 0.5% and a cell survival rate of over 83%. Furthermore, AOD's in vivo therapeutic efficacy against mouse subcutaneous abscess revealed its capability to restrain bacterial proliferation and reduce bacterial load, surpassing that of antibiotic lincomycin. These findings indicate AOD's potential for clinical usage.

Food-grade expression of nattokinase in Lactobacillus delbrueckii subsp. bulgaricus and its thrombolytic activity in vitro.

OBJECTIVES: To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. RESULTS: No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. CONCLUSIONS: This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.

Probiotics, prebiotics, and synbiotics regulate the intestinal microbiota differentially and restore the relative abundance of specific gut microorganisms.

Fermented milk is an effective carrier for probiotics, the consumption of which improves host health. The beneficial effects of probiotics, prebiotics, and synbiotics on gut dysbiosis have been reported previously. However, the way in which specific probiotics, prebiotics, and synbiotics regulate intestinal microbes remains unclear. Therefore, the probiotics Lactobacillus rhamnosus AS 1.2466 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 and the prebiotics xylooligosaccharide and red ginseng extracts were fed to mice to determine their effects on the intestinal microbiota. Then, mice were administered xylooligosaccharide and L. rhamnosus (synthesis) by gavage, and the number of L. rhamnosus was determined in the intestine at different times. The results show that probiotics and prebiotics can quickly reduce the Firmicutes/Bacteroidetes ratio, inhibit harmful bacteria (such as Klebsiella and Escherichia coli), and accelerate the recovery of beneficial intestinal microorganisms (such as Lactobacillus). In a complex intestinal microecology, different probiotics and prebiotics have different effects on specific intestinal microorganisms that cannot be recovered in the short term. In addition, after 20 d of intragastric xylooligosaccharide addition at 0.12 g/kg of body weight, L. rhamnosus colonization in the mouse ileum was 7.48 log cfu/mL, which was higher than in the low-dose group, prolonging colonization time and increasing the number of probiotics in the intestine. Therefore, this study demonstrated that probiotics and prebiotics can promote the balance of intestinal microbiota by regulating specific microbes in the intestine, and the effects of a suitable combination of synbiotics are beneficial, laying the foundation for the development of new dairy products rich in synbiotics.

Adhesion and Colonization of the Probiotic Lactobacillus rhamnosus Labeled by Dsred2 in Mouse Gut.

The health-promoting effects of the probiotic strain Lactobacillus rhamnosus are based on its adherence and colonization ability. However, little is known about its adhesion and colonization rates. Lactobacillus rhamnosus in mouse intestinal mucosa a mutant of the red fluorescence protein (RFP) DSred2 was used to tag L. rhamnosus to observe the adhesion and distribution of L. rhamnosus in mouse intestinal mucosa. A mutant of the red fluorescence protein (RFP) Dsred2 was used to tag L. rhamnosus to allow us to observe and distinguish it in the mouse intestine. Seven-week-old female BALB/c mice were fed once (at day 0) with an oral administration of the labeled L. rhamnosus, and the number of labeled bacteria was detected in different regions of the intestinal tract at 3 h and at day 1, 2, 3, 4, 5, 6, 7, and 15 after administration. The labeling process changed the morphology of L. rhamnosus, as it appeared after observation under the microscope, but did not change its basic probiotic properties in vitro. In vivo, labeled L. rhamnosus reached the colonization peak at the fourth day after gavage. From the distribution point of view, the number of colonization strains increased from the proximal to the distal small intestine (duodenum < jejunum < ileum) and the number of strains in the colon was less than the distal small intestine (ileum). The labeling protocol actually allowed the detection of the distribution and adhesion of this bacterium to the intestine, thus demonstrating that the health-promoting effects of this probiotic are satisfied. This study provides a scientific basis in the use of probiotics such as L. rhamnosus in functional foods.

Biological degradation of aflatoxin M1 by Bacillus pumilus E-1-1-1.

Aflatoxin M1 (AFM1 ) is a potent mycotoxin which causes serious health concerns in developing countries, where it is mainly found in milk, meat, and other foods. Biological detoxification is a promising method for eliminating AFM1 . The aim of this work was to search for AFM1 -degrading bacterial strains from animal waste, soil, and activated sludge. High-performance liquid chromatography and Fourier-transform infrared spectroscopy were used to analyze the AFM1 degradation products. A strain designated E-1-1-1 was obtained from African elephants feces, with the degradation ratio of AFM1 reaching 89.55% in 12 hr. Based on morphology, physiological and biochemical tests, and 16S rRNA gene sequence analysis, strain E-1-1-1 was identified as Bacillus pumilus. The culture supernatant of B. pumilus E-1-1-1 degraded AFM1 effectively, whereas the cells and cell extracts of B. pumilus E-1-1-1 were far less effective. Carbon and nitrogen sources had highly significant effects on the degradation of AFM1 by B. pumilus E-1-1-1. The AFM1 -degrading strain, B. pumilus E1-1-1, could have great potential in industrial applications.

Complete genome sequence of bacteriocin-producing Lactobacillus plantarum KLDS1.0391, a probiotic strain with gastrointestinal tract resistance and adhesion to the intestinal epithelial cells.

Lactobacillus plantarum KLDS1.0391 is a probiotic strain isolated from the traditional fermented dairy products and identified to produce bacteriocin against Gram-positive and Gram-negative bacteria. Previous studies showed that the strain has a high resistance to gastrointestinal stress and has a high adhesion ability to the intestinal epithelial cells (Caco-2). We reported the entire genome sequence of this strain, which contains a circular 2,886,607-bp chromosome and three circular plasmids. Genes, which are related to the biosynthesis of bacteriocins, the stress resistance to gastrointestinal tract environment and adhesive performance, were identified. Whole genome sequence of Lactobacillus plantarum KLDS1.0391 will be helpful for its applications in food industry.

Red Ginseng and Semen Coicis can improve the structure of gut microbiota and relieve the symptoms of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Many Chinese herbs are traditionally used as medicine to improve the functions of gastrointestinal tract. Some of these herbs are also promising agents for the improvement of the gut microbiota and the treatment of ulcerative colitis. MATERIALS AND METHODS: By screening seven traditional Chinese herbs, we found that Red Ginseng and Semen Coicis were the most effective in promoting the growth of probiotics including Lactobacillus and Bifidobacterium in vitro. We then evaluated the effects of Red Ginseng and Semen Coicis on the growth of the bacterial pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella spp.) in vitro. In in vivo experiment, we gavage administrated trinitro-benzene-sulfonic acid induced ulcerative colitis (UC) rats with Red Ginseng and Semen Coicis extracts. After two weeks treatment, we analyzed the structure of the gut microbiota and examined the UC symptoms by employing qPCR and animal pathology detection techniques. RESULTS: Both Red Ginseng and Semen Coicis promoted the growth of probiotics - Bifidobacterium and Lactobacillus in vitro. Red Ginseng also inhibited the growth of some pathogen strains. In vivo, Red Ginseng and Semen Coicis improved the structure of gut microbiota and relieved the symptoms of ulcerative colitis in vivo. Compared with Semen Coicis, Red Ginseng was more effective in relieving the symptoms of ulcerative colitis. CONCLUSIONS: Red Ginseng could promote the growth of probiotic bacteria in vitro. Red Ginseng and, to a lesser extent Semen Coicis, gave positive results in an experimental in vivo model for ulcerative colitis.

Increase of stress resistance in Lactococcus lactis via a novel food-grade vector expressing a shsp gene from Streptococcus thermophilus

The effects of the expression of a small heat shock protein (shsp) gene from Streptococcus thermophilus on stress resistance in Lactococcus lactis under different environmental stresses were investigated in this study. pMG36e-shsp, an expression vector, was first constructed by inserting a shsp open reading frame (ORF) cloned from S. thermophilus strain St-QC into pMG36e. Then, a food-grade expression vector, pMG-shsp, was generated by deleting the erythromycin resistance gene from pMG36e-shsp. The transformation rate of pMG-shsp was comparable to that of pMG36e-shsp when each of these two vectors was introduced into L. lactis. These results demonstrated that the shsp ORF could successfully used as a food-grade selection marker in both pMG-shsp and pMG36e-shsp. Furthermore, the growth characteristics were almost the same between L. lactis ML23 transformants harboring pMG36e or pMG-shsp. The survival rate of L. lactis ML23 expressing the shsp ORF were increased to 0.032%, 0.006%, 0.0027%, 0.03%, and 0.16% under the following environmental stresses: heat, acid, ethanol, bile salt and H2O2, respectively. These results indicated that the expression of the shsp gene in the food-grade vector pMG-shsp conferred resistance to environmental stresses without affecting the growth characteristics of L. lactis ML23.

Increase of stress resistance in Lactococcus lactis via a novel food-grade vector expressing a shsp gene from Streptococcus thermophilus.

The effects of the expression of a small heat shock protein (shsp) gene from Streptococcus thermophilus on stress resistance in Lactococcus lactis under different environmental stresses were investigated in this study. pMG36e-shsp, an expression vector, was first constructed by inserting a shsp open reading frame (ORF) cloned from S. thermophilus strain St-QC into pMG36e. Then, a food-grade expression vector, pMG-shsp, was generated by deleting the erythromycin resistance gene from pMG36e-shsp. The transformation rate of pMG-shsp was comparable to that of pMG36e-shsp when each of these two vectors was introduced into L. lactis. These results demonstrated that the shsp ORF could successfully used as a food-grade selection marker in both pMG-shsp and pMG36e-shsp. Furthermore, the growth characteristics were almost the same between L. lactis ML23 transformants harboring pMG36e or pMG-shsp. The survival rate of L. lactis ML23 expressing the shsp ORF were increased to 0.032%, 0.006%, 0.0027%, 0.03%, and 0.16% under the following environmental stresses: heat, acid, ethanol, bile salt and H2O2, respectively. These results indicated that the expression of the shsp gene in the food-grade vector pMG-shsp conferred resistance to environmental stresses without affecting the growth characteristics of L. lactis ML23.