An Evaluation of the Sensitivity and Applicability of a Droplet Digital Polymerase Chain Reaction Assay to Simultaneously Detect Pseudomonas aeruginosa and Pseudomonas fragi in Foods.

Achieving effective control over microbial contamination necessitates the precise and concurrent identification of numerous pathogens. As a common bacterium in the environment, Pseudomonas is rich in variety. It not only has pathogenic strains, but also spoilage bacteria that cause food spoilage. In this research, we devised a remarkably sensitive duplex droplet digital PCR (dddPCR) reaction system to simultaneously detect pathogenic Pseudomonas aeruginosa (P. aeruginosa) and spoilage Pseudomonas fragi (P. fragi). By employing comparative genomics, we identified four genes of P. fragi. Through a specific analysis, the RS22680 gene was selected as the detection target for P. fragi, and the lasR gene was chosen for P. aeruginosa, which were applied to construct a dddPCR reaction. In terms of specificity, sensitivity and anti-interference ability, the constructed dddPCR detection system was verified and analyzed. The assay showed excellent sensitivity and applicability, as evidenced by a limit of detection of 100 cfu/mL. When the concentration of natural background bacteria in milk or fresh meat was 100 times that of the target detection bacteria, the method was still capable of completing the absolute quantification. In the simulation of actual sample contamination, P. aeruginosa could be detected after 3 h of enrichment culture, and P. fragi could be detected after 6 h. The established dddPCR detection system exhibits exceptional performance, serving as a foundation for the simultaneous detection of various pathogenic bacteria in food products.

Recent advance in sesame allergens: Influence of food processing and their detection methods.

Sesame (Sesamum indicum L.) is a valuable oilseed crop with numerous nutritional benefits containing a diverse range of bioactive compounds. However, sesame is also considered an allergenic food that triggers various mild to severe adverse reactions (e.g., anaphylaxis). Strict dietary avoidance of sesame components is the best option to protect the sensitized consumers. Sesame or sesame-derived foods are always consumed after certain food processing operations, which would cause a considerable impact on the structure of sesame proteins, changing their sensitization capacity and detectability. In the review, the molecular structure properties, and immunological characteristics of the sesame allergens were described. Meanwhile, the influence of food processing techniques on sesame proteins and the relevant detection techniques used for the sesame allergens quantification are also emphasized critically. Hopefully, this review could provide valuable insight into the development and management for the new "Big Eight" sesame allergen in food industry.

Effects of dual modification by cationization and acetylation on the physicochemical and structural characteristics of glutinous rice starch.

In this research, the effects of cationization, acetylation and dual modification by cationization and acetylation on the physicochemical and structural characteristics of glutinous rice starches were investigated. The rapid viscosity analyzer revealed a substantial increased paste viscosity post modification. Particularly, for dually modified starch, the peak viscosity increased from 3071.67 to 4082.00 cP. The freeze-thaw stability substantially enhanced, with both single cationic and dually-modified starches standing out by exhibiting no water syneresis even at 21 freeze-thaw cycles, while native starch exhibited higher syneresis, up to 74.55 %. Both single cationization and cationization-acetylation destroyed the starch granules, characterized by the roughness and cracks. But, for single acetylation, there was no notable changes on granules' morphology. Fourier transform infrared spectroscopy exhibited notable shifts after modification, both acetylation and dual modification, resulting in a new peak at 1728 cm-1. 13C cross-polarization magic angle spinning nuclear magnetic resonance spectra displayed new peaks at 52-55 and 19-22 ppm following cationization and acetylation, respectively. These structural alterations indicate the successful incorporation of functional groups during modification. Overall, this study provides valuable insights for the industrial utilization of these three modified glutinous rice starches.

Analysis of Osmotic Tolerance, Physiological Characteristics, and Gene Expression of Salmonella enterica subsp. enterica Serotype Derby.

Salmonella is an important foodborne pathogen, and recent epidemiological studies have shown high infection rates of Salmonella enterica subsp. enterica serotype Derby (S.Derby) in poultry in western China and other regions. S.Derby presents increasing concerns with the development of resistance to hypertonic environments; however, there are few reports investigating the mechanism of resistance. Therefore, in this study, we examined hypertonic adaptation in S.Derby at the physiological and molecular levels. The K-B paper method, wiping glass bead method, crystal violet staining, and RT-PCR combined with comparative genomics analysis were employed to characterize virulence, drug resistance, biofilm formation, and changes in gene expression of genes related to hypertonic adaptation in S.Derby. Hypertonic-adapted S.Derby exhibited resistance to OXA, AMP, PEN, and CEP antibiotics, and biofilm-forming ability was 1.25 times that of nonadapted S.Derby. RT-PCR results showed that compared with nonadapted S.Derby, the expression of virulence-related genes in hypertonic-adapted S.Derby increased by 2-3 times, that of biofilm-related genes increased by 2-4 times, and that of OXA, AMP, PEN, and CEP-related drug resistance genes was relatively high. Four hypertonic tolerance-related genes (otsA, proV, proW, omsV) were preliminarily identified in S.Derby. The expression of proW was always relatively high in hypertonic-adapted S.Derby, the expression of otsA gradually became higher than that of proW with increasing time of osmotic stress, and the expression of proV and omsV was only high in non-hypertonic-adapted S.Derby.

Starch Modification with Molecular Transformation, Physicochemical Characteristics, and Industrial Usability: A State-of-the-Art Review.

Starch is a readily available and abundant source of biological raw materials and is widely used in the food, medical, and textile industries. However, native starch with insufficient functionality limits its utilization in the above applications; therefore, it is modified through various physical, chemical, enzymatic, genetic and multiple modifications. This review summarized the relationship between structural changes and functional properties of starch subjected to different modified methods, including hydrothermal treatment, microwave, pre-gelatinization, ball milling, ultrasonication, radiation, high hydrostatic pressure, supercritical CO2, oxidation, etherification, esterification, acid hydrolysis, enzymatic modification, genetic modification, and their combined modifications. A better understanding of these features has the potential to lead to starch-based products with targeted structures and optimized properties for specific applications.

Oxygen defects and S-scheme heterojunctions synergistically promote the photocatalytic hydrogen evolution activity and stability of WO2.72/Zn0.5Cd0.5S-DETA nanocomposites.

The study analyzed the impact of oxygen defects and S-scheme heterojunction on the performance and stability of WO2.72/Zn0.5Cd0.5S-DETA (WO/ZCS) nanocomposites photocatalysts for hydrogen evolution. Results showed that ZCS alone under visible light had good photocatalytic hydrogen evolution activity (1.762 mmol g-1h-1) and stability (79.5 % activity retention rate after seven cycles, 21 h). The WO3/ZCS nanocomposites with S-scheme heterojunction had better hydrogen evolution activity (2.287 mmol g-1h-1), but poor stability (41.6 % activity retention rate). The WO/ZCS nanocomposites with S-scheme heterojunction and oxygen defects showed excellent photocatalytic hydrogen evolution activity (3.94 mmol g-1h-1) and stability (89.7 % activity retention rate). The specific surface area measurement and ultraviolet-visible spectroscopy diffuse reflectance spectroscopy indicate that oxygen defects lead to larger specific surface area and improved light absorption, respectively. The charge density difference confirms the existence of the S-scheme heterojunction and the amount of charge transfer, which accelerates the separation of photogenerated electron-hole pairs and enhances the utilization efficiency of light and charge. This study offers a new approach using the synergistic impact of oxygen defects and S-scheme heterojunction to enhance the photocatalytic hydrogen evolution activity and stability.

Effects of C/N ratio on the growth and protein accumulation of heterotrophic Chlorella in broken rice hydrolysate.

BACKGROUND: Microalgae protein is considered as a sustainable alternative to animal protein in the future. Using waste for microalgal culture can upgrade low-value raw materials into high-value products, helping to offset the cost of microalgal protein production. In this study we explored the feasibility of using microalgae heterotrophic fermentation to convert broken rice hydrolysate (BRH) into protein. RESULTS: The results showed that the increase of BRH supplemental ratio was beneficial to the increase of biomass production but not beneficial to the increase of intracellular protein content. To further improve protein production, the effect of C/N ratio on intracellular protein accumulation was studied. It was found that low C/N ratio was beneficial to the synthesis of glutamate in microalgae cells, which in turn promoted the anabolism of other amino acids and further the protein. When the C/N ratio was 12:1, the biomass productivity and protein content could reach a higher level, which were 0.90 g/L/day and 61.56%, respectively. The obtained Chlorella vulgaris biomass was rich in essential amino acids (41.80%), the essential amino acid index was as high as 89.07, and the lysine content could reach up to 4.05 g/100 g. CONCLUSIONS: This study provides a theoretical basis and guidance for using Chlorella vulgaris as an industrial fermentation platform to convert broken rice into products with high nutritional value.

Mechanisms of promotion in the heterotrophic growth of Chlorella vulgaris by the combination of sodium acetate and hydrolysate of broken rice.

In order to reduce the culture cost and increase the growth rate of heterotrophic Chlorella vulgaris, the effects of hydrolysate of broken rice (HBR) combined with sodium acetate on its growth were evaluated. Results showed that the addition of 0.4 g/L of sodium acetate could stabilize the pH of the medium via the co-metabolism of acetate, ammonia and nitrate by Chlorella vulgaris. Meanwhile, isocitrate lyase activity increased threefold, which further promoted the glyoxylate cycle and the citric acid cycle, which finally provided more energy and metabolic precursors for cell growth. The biomass production (5.04 g/L), biomass productivity (1.65 g/L/day) and protein content (64.14 %) were 1.56, 1.81 and 1.77 times higher than the glucose group. This study demonstrated that HBR combined with sodium acetate could effectively promote the heterotrophic metabolism of microalgae, which provided scientific basis and guidance for industrial production of high-value products using Chlorella vulgaris as a fermentation platform.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Changes in the multi-scale structure and physicochemical properties of starch during potato growth.

BACKGROUND: Growth stage contributes critically to the physicochemical properties of starches, which make achieving desired functional properties by controlling the growth period possible. Thus, this study investigated the changes in multiscale structure and physicochemical properties of potatoes starches harvested at different growth stages. RESULTS: The amylose and phosphate content varied over the growth period, with the ranges 2.756-2.998 g kg-1 and 0.0058-0.0077 g kg-1 , respectively. The starch granules were round or oval, and the size increased with growth. X-Ray diffraction indicated the B-type crystalline structure of samples. Time-dependent changes in crystallinity were observed. The weight-average molecular weight (Mw ) showed a tendency to decrease first and then increase, and presented the lowest Mw (1.105 × 108 g mol-1 ) at 35 days. A higher proportion of long chains were noted in starch from earlier harvested potatoes than that in later harvested ones. Differential scanning calorimetry revealed that starch gelatinization temperature decreased, and gelatinization enthalpy decreased from 16.39 to 14.89 J g-1 . All samples possessed weak elastic gel-like structure, and starches harvested at early stage possessed highest viscosity and stronger gel behaviour. Resistant starch showed a decreasing trend on the whole, and presented highest value (10.69%) at earliest harvest time. Starch from the potatoes harvested at 35 days after tuberization exhibited excellent light transmittance (up to 62.47%). CONCLUSION: Potato starches harvested at different growth period presented extremely different structures and physicochemical properties. The results will provide fundamental data in terms of changes of potato starch during growth which will affect the choice of harvest time. © 2021 Society of Chemical Industry.

Propidium monoazide real-time PCR amplification for viable Salmonella species and Salmonella Heidelberg in pork.

Salmonella enterica serovar Heidelberg causes foodborne infections and is a major threat to the food chain and public health. In this study, we aimed to develop a rapid molecular typing approach to identify Salmonella enterica serovar Heidelberg. Using comparative genomics, four serovar-specific gene fragments were identified, and a real-time polymerase chain reaction (PCR) combined with a propidium monoazide (PMA) pretreatment method was developed for simultaneous detection of viable Salmonella sp. (invA) and Salmonella Heidelberg (SeHA_C3258). The assay showed 100% specificity for all strains tested. The assay was able to distinguish effectively viable or dead cells with the PMA. The detection limit was 2.4 CFU/mL following 6 h of incubation in enrichment Luria-Bertani medium, and the assay could detect 1.7 × 102 CFU/mL in the presence of pork background flora. In artificially contaminated pork, real-time PCR detected inoculum levels of 1.15 CFU/25 g of pork after a 6 h enrichment. Thus, our findings indicated that this comparative genomics approach could be used to screen for serovar-specific fragments and that real-time PCR with PMA was a simple and reliable method for detecting viability of Salmonella species and Salmonella Heidelberg.

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10-1 CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5 × 103 CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

Amino acid decarboxylase-dependent acid tolerance, selected phenotypic, and virulence gene expression responses of Salmonella enterica serovar Heidelberg.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the pathogens most frequently detected in recent years in food products from animals; here, we examine the acid tolerance of this strain. Mild acid (pH5.5-5.0) induced a strong acid resistance in S. Heidelberg; the induced acid resistance improved within 0.5-6h and resulted in >95% cell survival following challenge in pH3.0 medium. Addition of lysine or arginine to pH2.5 acid challenge medium significantly improved the survival of S. Heidelberg; lysine induced the largest increase in survival. The lysine and arginine decarboxylase-related genes (i.e., cadA, cadB, adiA, and adiY) are acid induced genes, and they play an important role in S. Heidelberg acid resistance. RT-PCR showed that the genes expression levels increased as acid adaptation pH decreased. The increased expression was maintained for at least 4h during adaptation. Moreover, acid adaptation may have increased the production of cellulose and swimming in S. Heidelberg; pH5.5 acid-adapted cells showed a red, dry, and rough morphotype on Congo red plates. The transcriptional levels of Enterotoxin gene (stn) increased three times following acid adaptation; however, the expression of Salmonella pathogenicity island 1 virulence genes significantly decreased.

Development of a PCR test system for specific detection of Salmonella Paratyphi B in foods.

Salmonella enterica serotype Paratyphi B is a globally distributed human-specific pathogen causing paratyphoid fever. The aim of this study was to develop a rapid and reliable polymerase chain reaction (PCR) assay for its detection in food. The SPAB_01124 gene was found to be unique to S. Paratyphi B using comparative genomics. Primers for fragments of the SPAB_01124 gene and the Salmonella-specific invA gene were used in combination to establish a multiplex PCR assay that showed 100% specificity across 45 Salmonella strains (representing 34 serotypes) and 18 non-Salmonella strains. The detection limit was 2.2 CFU mL(-1) of S. Paratyphi B after 12-h enrichment in pure culture. It was shown that co-culture with S. Typhimurium or Escherichia coli up to concentrations of 3.6 × 10(5)  CFU and 3.3 × 10(4)  CFU, respectively, did not interfere with PCR detection of S. Paratyphi B. In artificially contaminated milk, the assay could detect as few as 62 CFU mL(-1) after 8 h of enrichment. In conclusion, comparative genomics was found to be an efficient approach to the mining of pathogen-specific target genes, and the PCR assay that was developed from this provided a rapid, specific, and sensitive method for detection of S. Paratyphi B.

Detection of Salmonella enterica serovar Dublin by polymerase chain reaction in multiplex format.

S. Dublin has caused widespread concerns in cattle produce. Using a comparative genomic method, two specific targets like SeD_A1118 and SeD_A2283 for S. Dublin identification were firstly obtained. An efficient multiplex PCR for S. Dublin detection based on the two novel specific genes and invA was therefore developed.

Mining and evaluation of new specific molecular targets for the PCR detection of Salmonella spp. genome.

The purpose of this study is to find new molecular targets for the detection of Salmonella. With the online BLAST Program, we compared homology of genomic sequences and specificity in GenBank among Salmonella serovars and non-Salmonella strains and found 98 Salmonella specific target sequences. We selected 33 target sequences of Gene ID from 3335000 to 3337003 for the specificity evaluation, and finally 8 specific fragments screened out, they are 3334138, 3335583, 3335471, 3335211, 3335068, 3336466, 3336736 and 3336998. Primer SC8 of gene 3335583 and SC9 of gene 3335471 were the best in specificity and sensitivity among these primers. The detection sensitivity of Primer SC9 was 1.23 fg/µl for DNA templates and 720 cfu/ml for whole cells, while primer SC8's was 12.3 fg/µl and 720 cfu/ml, respectively. Salmonella could be detected successfully by the PCR method developed in this study after 8 h enrichment when the milk samples were artificially contaminated by this organism at 7 cfu per 10 ml milk.