Postharvest quality maintenance of wax apple and guava fruits by use of a fermented broth of an ε-poly-l-lysine-producing Streptomyces strain.

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial polymer with significant inhibitory activity against a broad spectrum of microorganisms, and nowadays used widely as a preservative in the food industry. In the present study, ε-PL broth was obtained from Streptomyces ahygroscopicus GIM8 fermentation in a nutrient-limited liquid medium. The in vitro antifungal activity of the broth against fruit pathogens Penicillium expansum and Colletotrichum gloeosporioides was investigated, and its usage for postharvest storage of two highly perishable fruits wax apple and guava was evaluated. Results showed that ε-PL concentration in the broth reached 0.61 g/L, and the nutrition level of the broth was low. The antifungal activity of ε-PL broth was comparable to that of the aqueous solution of ε-PL under the same concentration. Immersion with the diluted broth (200 mg/L ε-PL) markedly delayed the decline in the quality of postharvest wax apple and guava fruits during storage, and the decay incidences were also greatly decreased as compared to their respective controls (distilled water immersion). A further investigation demonstrated that the ε-PL broth immersion induced an increase in the activity of defense-related enzymes peroxidase and polyphenol oxidase in the two fruits during storage. The present study proved that the fermentation broth of ε-PL could be used as a promising alternative to high purity ε-PL and synthetic fungicides for preserving fruits at postharvest stage.

Early Death and Survival of Patients With Acute Promyelocytic Leukemia in ATRA Plus Arsenic Era: A Population-Based Study.

Most randomized trials for acute promyelocytic leukemia (APL) have investigated highly selected patients under idealized conditions, and the findings need to be validated in the real world. We conducted a population-based study of all APL patients in Zhejiang Province, China, with a total population of 82 million people, to assess the generalization of all-trans retinoic acid (ATRA) and arsenic as front-line treatment. The outcomes of APL patients were also analyzed. Between January 2015 and December 2019, 1,233 eligible patients were included in the final analysis. The rate of ATRA and arsenic as front-line treatment increased steadily from 66.2% in 2015 to 83.3% in 2019, with no difference among the size of the center (≥5 or <5 patients per year, p = 0.12) or age (≥60 or <60 years, p = 0.35). The early death (ED) rate, defined as death within 30 days after diagnosis, was 8.2%, and the 3-year overall survival (OS) was 87.9% in the whole patient population. Age (≥60 years) and white blood cell count (>10 × 109/L) were independent risk factors for ED and OS in the multivariate analysis. This population-based study showed that ATRA and arsenic as front-line treatment are widely used under real-world conditions and yield a low ED rate and a high survival rate, which mimic the results from clinical trials, thereby supporting the wider application of APL guidelines in the future.

Amplified electrochemical antibiotic aptasensing based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework.

A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.

Identification of the Potential Biological Preservative Tetramycin A-Producing Strain and Enhancing Its Production.

The aim of this study was to develop a potential microbial preservative to prevent the growth of fungi in food. The isolate ZC-G-5 showed strong antifungal activity against food spoilage fungi and Streptomyces albulus was identified on the basis of morphologic, culture, and 16S rDNA sequence analyses. The active metabolite was elucidated as tetramycin A (TMA) through spectroscopic techniques, including HR-ESI-MS, 1D-NMR, and 2D-NMR. An antifungal activity assay revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TMA were 1.50-2.50 and 3.00-5.00 µg/ml, respectively. In situ antifungal activity analyses demonstrated that 90.0 µg/ml of TMA could inhibit the growth of fungi for over 14 days. In order to enhance TMA production, the high-yield mutant strain YB101 was screened, based on the isolate ZC-G-5, using a high-throughput screening method. The best metabolic precursor was selected during fermentation, when the concentration of glycerol was 8% (v/v) in Gauze's broth medium to cultivate the mutant strain YB101; the concentration of TMA could be increased to 960.0 µg/ml, compared with the original isolate ZC-G-5, where the concentration of the TMA was only 225.0 µg/ml. Our study may contribute to the application of S. albulus and its active metabolite as a potential bio-preservative in the food industry.

Classification and fatty acid composition analysis of Cronobacter spp. isolated from powdered infant formula in China.

This study aimed to classify a collection of Enterobacter sakazakii (E. sakazakii) strains previously identified from powdered infant formula (PIF) to species level by recN gene sequencing and biochemical testing to determine the distribution of Cronobacter species in China and investigate the strain diversity by cellular fatty acid (CFA) analysis. Of 24 E. sakazakii isolates, 23 were identified as C. sakazakii and one was C. malonaticus. The 23 C. sakazakii isolates showed the same CFA profiles. The C. malonaticus isolate was discriminated from the C. sakazakii isolates by the significant difference in the amounts of C12:0, C14:0, and C17:0 cyclo acids. These results showed that C. sakazakii and C. malonaticus were the common Cronobacter species distributed in PIF in China and that the isolates of the two species exhibited different CFA profiles. These findings are of value for epidemiological investigations and provide an alternative method for confirming various Cronobacter spp.

Investigation on the effects of ϵ-poly-L-lysine on a producing strain Streptomyces ahygroscopicus GIM8, for better understanding its biosynthesis.

ϵ-Poly-L-lysine (ϵ-PL) is an L-lysine homopolymer with strong antimicrobial activity, which is generally produced by Streptomyces strains. ϵ-PL is only produced under acidic conditions in liquid culture, and to improve the current understanding of ϵ-PL biosynthesis, the present study was undertaken to investigate the effects of ϵ-PL on its producer Streptomyces ahygroscopicus GIM8, under acidic and neutral conditions. The results indicated that a neutral pH favored ϵ-PL adsorption onto the cells, whereas minimal adsorption occurred at pH 4.0, the maximum pH for ϵ-PL production. At pH 7.0, small amounts of ϵ-PL caused considerable ATP leakage from the cells, which showed increased membrane permeability. Conversely, ATP leakage was inhibited by ϵ-PL at pH 4.0. Transmission electron microscopy investigation indicated that the cytoplasmic membrane was the primary site of ϵ-PL activity at pH 7.0, and that cell shape was maintained. Metabolic activity profiles revealed that ϵ-PL decreased cellular metabolic activity at a relatively low rate at pH 7.0. However, the toxic effect was significantly enhanced at pH 4.0. Based on these data, a mechanism for the effect of ϵ-PL on ϵ-PL-producing cells under neutral and acidic conditions is proposed. Additionally, acidic conditions may potentially be required for ϵ-PL biosynthesis in liquid culture because low pH can increase membrane permeability and prevent binding of ϵ-PL onto cells, both of which favor the secretion of the ϵ-PL produced by the cells into the broth. This research contributes to the current understanding of ϵ-PL biosynthesis.

Efficient production of ε-poly-L-lysine by Streptomyces ahygroscopicus using one-stage pH control fed-batch fermentation coupled with nutrient feeding.

ε-Poly-L-lysine (ε-PL) is a homopolymer of L-lysine molecules connected between the ε amino and alpha carboxyl groups. This polymer is currently used as a natural preservative in food. Insufficient biomass is a major problem in ε-PL fermentation. Here, to improve cell growth and ε-PL productivity, various nitrogen-rich nutrients were supplemented into flask cultures after 16 h cultivation, marking the onset of ε-PL biosynthesis. Yeast extract, soybean powder, corn powder, and beef extract significantly improved cell growth. In terms of ε-PL productivity, yeast extract at 0.5% (w/v) gave the maximum yield (2.24 g/l), 115.4% higher than the control (1.04 g/l), followed by soybean powder (1.86 g/l) at 1% (w/v) and corn powder (1.72 g/l) at 1% (w/v). However, supplementation with beef extract inhibited ε-PL production. The optimal time for supplementation for all nutrients examined was at 16 h cultivation. The kinetics of yeast-extract-supplemented cultures showed enhanced cell growth and production duration. Thus, the most commonly used two-stage pH control fed-batch fermentation method was modified by omitting the pH 5.0-controlled period, and coupling the procedure with nutrient feeding in the pH 3.9-controlled phase. Using this process, by continuously feeding 0.5 g/h of yeast extract, soybean powder, or corn powder into cultures in a 30 L fermenter, the final ε-PL titer reached 28.2 g/l, 23.7 g/l, and 21.4 g/l, respectively, 91.8%, 61.2%, and 45.6% higher than that of the control (14.7 g/l). This describes a promising option for the mass production of ε-PL.

[Isolation and identification of Cronobacter (Enterobacter sakazakii) strains from food].

OBJECTIVE: This study aimed to detect and quantify Cronobacter in 300 powdered milk samples and 50 non-powdered milk samples. Totally, 24 Cronobacter (formerly Enterobacter sakazakii) strains isolated from powdered milk and other foods were identified and confirmed. METHODS: Cronobacter strains were detected quantitatively using most probable number (MPN) method and molecular detection method. We identified 24 Cronobacter strains using biochemical patterns, including indole production and dulcitol, malonate, melezitose, turanose, and myo-Inositol utilization. Of the 24 strains, their 16S rRNA genes were sequenced, and constructed phylogenetic tree by N-J (Neighbour-Joining) with the 16S rRNA gene sequences of 17 identified Cronobacter strains and 10 non-Cronobacter strains. RESULTS: Quantitative detection showed that Cronobacter strains were detected in 23 out of 350 samples yielding 6.6% detection rate. Twenty-four Cronobacter strains were isolated from 23 samples and the Cronobacter was more than 100 MPN/100g in 4 samples out of 23 samples. The 24 Cronobacter spp. isolates strains were identified and confirmed, including 19 Cronobacter sakazakii strains, 2 C. malonaticus strains, 2 C. dubliensis subsp. lactaridi strains, and 1 C. muytjensii strain. CONCLUSION: The combination of molecular detection method and most probable number (MPN) method could be suitable for the detection of Cronobacter in powdered milk, with low rate of contamination and high demand of quantitative detection. 24 isolated strains were confirmed and identified by biochemical patterns and molecular technology, and C. sakazakii could be the dominant species. The problem of Cronobacter in powdered milk should be a hidden danger to nurseling, and should catch the government and consumer's attention.

Sequencing of the grxB gene of Cronobacter spp. and the development of a PCR assay for its identification.

Cronobacter spp. (formerly Enterobacter sakazakii), a foodborne pathogen linked to powdered infant formula, is a rare cause of invasive infection with a high mortality rate in neonates. In this study, the Cronobacter sakazakii ATCC 29544 and C. muytjensii ATCC 51329 glutaredoxin 2 (grxB) genes were cloned and sequenced. Based on the unique regions of the Cronobacter grxB genes, two primers were synthesized to develop and optimize a Cronobacter-specific polymerase chain reaction (PCR) method. The PCR assay amplified a 378-bp DNA product from all positive controls, which are composed of 45 strains of Cronobacter spp., but not from any of 45 non-Cronobacter bacterial strains. The detection limits of this method are 10(4) colony-forming units (CFU)/mL of Cronobacter spp. in infant formula directly and 10(0) CFU/mL after an 8-h enrichment step. In summary, we have developed a PCR assay based on the grxB sequence. Combined with enrichment culturing, this technique offers a rapid and sensitive method for the detection of Cronobacter spp.

Enhanced epsilon-poly-L-lysine production from Streptomyces ahygroscopicus by a combination of cell immobilization and in situ adsorption.

Epsilon-poly-L-lysine (epsilon-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce epsilon-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more epsilon-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced 0.54 +/- 0.1 g/l epsilon-PL, which significantly increased to 3.64 +/- 0.32 g/l after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded 2.73 +/- 0.26 g/l of epsilon-PL, an increase from 0.82 +/- 0.08 g/l. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an epsilon-PL total amount of 8.05 +/- 0.84 g/l was obtained. The proposed combination method offers promising perspectives for epsilon-PL production.

Production of ε-poly-L-lysine by Streptomyces sp. using resin-based, in situ product removal.

Resin-based, in situ product removal (ISPR) was used to increase production of ε-poly-L-lysine (PL) by Streptomyces sp. GIM8. D152 resin was selected over Amberlite IRC-50, Amberlite IRC-76 and Amberlite IR-120 to develop ISPR using adsorption capacity and desorption ratio as bases. The yield of PL in response to external PL was unaffected in shake-flask culture; however, the production of PL increased to 2.9 from 0.8 g l(-1) shake-flasks using ISPR. In a 5 l fermentor, 23.4 g PL l(-1) was achieved compared to 3.76 g PL l(-1), in the controls by attaching two bags of D152 resin to the probes and baffles of the fermentor.