Recent advances in understanding the control of disinfectant-resistant biofilms by hurdle technology in the food industry.

Modern food processing environment provides an ideal condition for biofilms formation by foodborne and spoilage microorganisms on different food contact surfaces. It is widely acknowledged that biofilm has become a serious problem in the food industry, as the biofilm growth mode induces microbial resistance to chemical disinfection. The persistence of biofilms after cleaning and disinfection procedures may result in foodborne illness and food spoilage, emphasizing the importance of preventing biofilms in food production facilities. The use of conventional disinfection technologies alone may not help to achieve the goal of producing safe food products with high quality. Hurdle technology provides a great option for the effective control of biofilms formed on food contact surfaces. Thus, a better understanding of biofilm behavior in response to different disinfectants, as well as seeking potential hurdle technologies to control biofilms are essential. In this review, we discuss the factors that influence the efficiency of disinfectants, and elaborate possible mechanisms which are behind the apparent high antimicrobial resistance of biofilms, and as well as mechanisms which are involved in effective hurdle technologies to control biofilms.

Establishment and evaluation of a method for efficient screening of Clostridium butyricum.

At present, the traditional methods for the screening of Clostridium butyricum are not sufficiently selective and efficient. Therefore, it is necessary to establish a targeted and efficient screening method for the detection of C. butyricum. Bioinformatics was used in this study to find C. butyricum specific genes, and species-specific primers were designed based on the conserved regions of the targeted genes, followed by optimization of the PCR conditions. Methodological evaluation was carried out, and the results were compared with the traditional screening method based on Trypticase Sulfite Neomycin (TSN) selective medium. A high-efficiency PCR screening method, targeting C. butyricum species-specific primers, was established. The method was confirmed to have high specificity and sensitivity towards C. butyricum cut-off CFU 103. Compared with the traditional method, the screening success rate of C. butyricum strains increased from 0.61 to 81.91%. The PCR screening method could quickly and accurately detect C. butyricum in samples and dramatically improve screening efficiency.

Interspecies Interactions in Dual-Species Biofilms Formed by Psychrotrophic Bacteria and the Tolerance of Sessile Communities to Disinfectants.

ABSTRACT: Biofilms on the surface of food processing equipment act as potential reservoirs of microbial contamination. Bacterial interactions are believed to play key roles in both biofilm formation and antimicrobial tolerance. In this study, Aeromonas hydrophila, Chryseobacterium oncorhynchi, and Pseudomonas libanensis, which were previously isolated from Chinese raw milk samples, were selected to establish two dual-species biofilm models (P. libanensis plus A. hydrophila and P. libanensis plus C. oncorhynchi) on stainless steel at 7°C. Subsequently, three disinfectants, hydrogen peroxide (100 ppm), peracetic acid (100 ppm), and sodium hypochlorite (100 ppm), were used to treat the developed sessile communities for 10 min. Structural changes after exposure to disinfectants were analyzed with confocal laser scanning microscopy. The cell numbers of both A. hydrophila and C. oncorhynchi recovered from surfaces increased when grown as dual species biofilms with P. libanensis. Dual-species biofilms were more tolerant of disinfectants than were each single-species biofilm. Peracetic acid was the most effective disinfectant for removing biofilms, followed by hydrogen peroxide and sodium hypochlorite. The results expand the knowledge of mixed-species biofilms formed by psychrotrophic bacteria and will be helpful for developing effective strategies to eliminate bacterial mixed-species biofilms.

Preparation screening, production optimization and characterization of exopolysaccharides produced by Lactobacillus sanfranciscensis Ls-1001 isolated from Chinese traditional sourdough.

The present study focused on preparation screening, production optimization and characterization of exopolysaccharides (EPS) produced by Lactobacillus sanfranciscensis isolated from a Chinese traditional sourdough sample. In addition, in vitro antioxidant activity of pure EPS was determined. A total of 51 strains of L. sanfranciscensis were screened, and L. sanfranciscensis Ls-1001 was proved as the most abundant crude EPS producing strain, which was selected for further EPS optimization and characterization. The optimized conditions resulted in around 31% increase in EPS production (final yield: 249.30 mg/L) and included: maltose 25 g/L, yeast peptone 10.24 g/L and fresh yeast extractives (FYE) 12.92 mL/L. HPLC analysis showed that pure EPS contained only glucose monomers, indicating that it was a kind of homopolysaccharides. FTIR spectroscopy revealed the presence of hydroxyl and carbonyl groups. NMR analysis confirmed that EPS contained α-glycosidic bond and pyranose residue. The EPS showed strong in vitro antioxidant activity.

Insights into Psychrotrophic Bacteria in Raw Milk: A Review.

HIGHLIGHTS: Levels of psychrotrophic bacteria in raw milk are affected by to habitats and farm hygiene. Biofilms formed by psychrotrophic bacteria are persistent sources of contamination. Heat-stable enzymes produced by psychrotrophic bacteria compromise product quality. Various strategies are available for controlling dairy spoilage caused by psychrotrophic bacteria.

Identification of Key Aroma Compounds in Type I Sourdough-Based Chinese Steamed Bread: Application of Untargeted Metabolomics Analysisp.

Untargeted metabolomics is a valuable tool to analyze metabolite profiles or aroma fingerprints of different food products. However, less attention has been paid to determining the aroma characteristics of Chinese steamed breads (CSBs) by using this approach. The aim of this work was to evaluate the key aroma compounds and their potential generation pathway in Chinese steamed bread produced with type I sourdough by a metabolomics approach. Based on the aroma characteristics analysis, CSBs produced with type I sourdough and baker's yeast were clearly distinguishable by principal component analysis (PCA) scores plot. A total of 13 compounds in sourdough-based steamed breads were given the status of discriminant markers through the untargeted metabolomics analysis. According to the odor activity values (OAVs) of discriminant aroma markers, ethyl acetate (fruity), ethyl lactate (caramel-like), hexyl acetate (fruity), (E)-2-nonenal (fatty) and 2-pentylfuran (fruity) were validated as the key volatile compounds in the breads produced with type I sourdough as compared to the baker's yeast leavened steamed bread. The metabolite analysis in proofed dough indicated that esters are mainly generated by the reaction between acid and alcohol during steaming, and aldehydes are derived from the oxidation of palmitoleic acid and linoleic acid during proofing and steaming.

Insights into Bacterial Milk Spoilage with Particular Emphasis on the Roles of Heat-Stable Enzymes, Biofilms, and Quorum Sensing.

Milk spoilage caused by psychrotrophic bacteria and their heat-stable enzymes is a serious challenge for the dairy industry. In many studies, spoilage has been explored based on the simplistic view of undesirable enzymes produced by planktonic cells. Recently, biofilms and quorum sensing (QS) have been suggested as important factors in the deterioration of milk, which opens new avenues for investigation of the processes and challenges. Production and heat stability of enzymes are enhanced in biofilms, mainly because of inherent differences in physiological states and protective shielding by extracellular polymeric substances. QS plays a key role in modulating expression of hydrolytic enzymes and biofilm formation. To date, few studies have been conducted to investigate the complex interplays of enzyme production, biofilm formation, and QS. This review provides novel insights into milk spoilage with particular emphasis on the roles of biofilms and QS and summarizes potential effective strategies for controlling the spoilage of milk.

Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes.

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive ß-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.

Analysis of bacterial diversity and biogenic amines content during the fermentation processing of stinky tofu.

Fermented foods may confer several benefits to human health and play an important role in a healthy and balanced diet. Stinky tofu is an important traditional fermented soy product in Asia. In this study, a comprehensive analysis of bacterial diversity in terms of rarefaction abundance, rank abundance, alpha diversity, and principal coordinates analysis was conducted using next-generation sequencing. The results obtained were further verified by culture-dependent methods. In addition, eight biogenic amines were also quantified to determine their levels in stinky tofu during conventional production. Our study revealed that lactobacillus was the major lactic acid bacteria throughout the fermentation process. Further, some potentially harmful bacterial belonging to the genera Halomonas and Solobacterium, were also found existing in the stinky tofu. In case of biogenic amines, the concentration of putrescine, cadaverine and histamine finally increased to high levels while spermine disappeared after the fermentation was completed. After the soaking step, putrescine, tryptamine and ß-phenethylamine increased sharply. This work provided an improved understanding of microbes and biogenic amines content associated with the traditionally consumed fermented food. However, there is a need to understand microbial interactions related to biogenic amines during the fermentation, and the origin and survival strategies of two important pathogens detected in our study.

Predominant yeasts in Chinese traditional sourdough and their influence on aroma formation in Chinese steamed bread.

A total of 105 yeast isolates was obtained from 15 sourdough samples collected from different regions in China and subjected to random amplified polymorphic DNA (RAPD) analysis. Six species were identified including Pichia membranifaciens, which has not previously been reported in Chinese sourdoughs. Different species of yeast were used in single-culture fermentation to make Chinese steamed bread (CSB). The volatiles of the CSB were captured by solid-phase microextraction method, separated and identified by gas chromatography-mass spectrometry. In total, 41 volatile compounds were found in all the steamed breads. All CSBs showed a similar volatile profile; however, significant differences in the quantity of some volatile compounds were seen among the CSB fermented by different yeast species. A partial least squares discriminant analysis showed that the CSBs could be separated by their characteristic volatile profiles. The study suggested that the aromatic properties of CSB are determined by the yeast used.

Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders.

Biofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3logCFU/cm2, respectively. Number of cells recovered from biofilms on 1cm2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk. Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasize strain and substrate dependence.

Phenotypic and genetic heterogeneity within biofilms with particular emphasis on persistence and antimicrobial tolerance.

Phenotypic changes or phase variation within biofilms is an important feature of bacterial dormant life. Enhanced resistance to antimicrobials is one of the distinct features displayed by a fraction of cells within biofilms. It is believed that persisters are mainly responsible for this phenotypic heterogeneity. However, there is still an unresolved debate on the formation of persisters. In this short review, we highlight all known genomic and proteomic changes encountered by bacterial cells within biofilms. We have also described all phenotypic changes displayed by bacterial cells within biofilms with particular emphasis on enhanced antimicrobial tolerance of biofilms with particular reference to persisters. In addition, all currently known models of persistence have been succinctly discussed.

Insights into the microbial diversity and community dynamics of Chinese traditional fermented foods from using high-throughput sequencing approaches.

Chinese traditional fermented foods have a very long history dating back thousands of years and have become an indispensable part of Chinese dietary culture. A plethora of research has been conducted to unravel the composition and dynamics of microbial consortia associated with Chinese traditional fermented foods using culture-dependent as well as culture-independent methods, like different high-throughput sequencing (HTS) techniques. These HTS techniques enable us to understand the relationship between a food product and its microbes to a greater extent than ever before. Considering the importance of Chinese traditional fermented products, the objective of this paper is to review the diversity and dynamics of microbiota in Chinese traditional fermented foods revealed by HTS approaches.

New mechanistic insights into the motile-to-sessile switch in various bacteria with particular emphasis on Bacillus subtilis and Pseudomonas aeruginosa: a review.

A biofilm is a complex assemblage of microbial communities adhered to a biotic or an abiotic surface which is embedded within a self-produced matrix of extracellular polymeric substances. Many transcriptional regulators play a role in triggering a motile-sessile switch and in consequently producing the biofilm matrix. This review is aimed at highlighting the role of two nucleotide signaling molecules (c-di-GMP and c-di-AMP), toxin antitoxin modules and a novel transcriptional regulator BolA in biofilm formation in various bacteria. In addition, it highlights the common themes that have appeared in recent research regarding the key regulatory components and signal transduction pathways that help Bacillus subtilis and Pseudomonas aeruginosa to acquire the biofilm mode of life.

A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough.

Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB still remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distillation-extraction (SDE), and purge and trap (P&T). All samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies.

The heat resistance and spoilage potential of aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders.

The propensity for aerobic bacilli and allied genera to produce highly heat-resistant spores and thermally stable spoilage enzymes are major bacteriological issues faced by the dairy industry. Most of the enzymes are able to survive any heat treatment applied during the manufacture of milk powders and have the potential to remain active in milk powders and other dairy products during storage, and may explain some of the sensory and functionality defects reported in dairy products. Despite many reports on the occurrence of spore-forming bacteria in dairy products, knowledge about food quality related properties of many aerobic sporeformers is still scarce. Therefore, the aim of this study was to determine thermal resistance and spoilage potential of a large pool of mesophilic and thermophilic sporeformers, representing 738 isolates and 31 different RAPD groups, recently isolated from Chinese milk powders. Spore formers producing highly heat resistant spores (surviving 125°C for 30min) included 2 thermophiles (Geobacillus thermoleovorans group and Geobacillus stearothermophilus) and one mesophilic species (Brevibacillus brevis). Paenibacillus macerans showed the highest proteolytic activity followed by members of the Bacillus cereus group, Br. brevis, Bacillus subtilis, G. thermoleovorans group and Virgibacillus proomii. The highest lipase producing strains belonged to Bacillus licheniformis. Phospholipase activity was only shown by members of the B. cereus group and Brevibacillus parabrevis. Ten strains showed positive ß-galactosidase activity, while, 4 strains showed positive haemolytic activity. B. licheniformis strains, despite belonging to one RAPD group or sub-group showed markedly different phenotypic characters which support the previous findings of heterogeneity in RAPD-based B. licheniformis groups. The results of this study will broaden the knowledge about the spoilage potential and thermal resistance of many strains of dairy origin.

Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Enzymatic Hydrolysates of Razor Clam Sinonovacula constricta.

Angiotensin I-converting enzyme (ACE) inhibitory activity of razor clam hydrolysates produced using five proteases, namely, pepsin, trypsin, alcalase, flavourzyme and proteases from Actinomucor elegans T3 was investigated. Flavourzyme hydrolysate showed the highest level of degree of hydrolysis (DH) (45.87%) followed by A. elegans T3 proteases hydrolysate (37.84%) and alcalase (30.55%). The A. elegans T3 proteases was observed to be more effective in generating small peptides with ACE-inhibitory activity. The 3 kDa membrane permeate of A. elegans T3 proteases hydrolysate showed the highest ACE-inhibitory activity with an IC50 of 0.79 mg/mL. After chromatographic separation by Sephadex G-15 gel filtration and reverse phase-high performance liquid chromatography, the potent fraction was subjected to MALDI/TOF-TOF MS/MS for identification. A novel ACE-inhibitory peptide (VQY) was identified exhibiting an IC50 of 9.8 µM. The inhibitory kinetics investigation by Lineweaver-Burk plots demonstrated that the peptide acts as a competitive ACE inhibitor. The razor clam hydrolysate obtained by A. elegans T3 proteases could serve as a source of functional peptides with ACE-inhibitory activity for physiological benefits.

A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China.

Aerobic spore forming bacteria are potential milk powder contaminants and are viewed as indicators of poor quality. A total of 738 bacteria, including both mesophilic and thermophilic, isolated from twenty-five powdered milk samples representative of three types of milk powders in China were analyzed based on the random amplified polymorphic DNA (RAPD) protocol to provide insight into species diversity. Bacillus licheniformis was found to be the most prevalent bacterium with greatest diversity (~43% of the total isolates) followed by Geobacillus stearothermophilus (~21% of the total isolates). Anoxybacillus flavithermus represented only 8.5% of the total profiles. Interestingly, actinomycetes represented a major group of the isolates with the predominance of Laceyella sacchari followed by Thermoactinomyces vulgaris, altogether comprising of 7.3% of the total isolates. Out of the nineteen separate bacterial species (except five unidentified groups) recovered and identified from milk powders, twelve proved to belong to novel or previously unreported species in milk powders. Assessment and characterization of the harmful effects caused by this particular micro-flora on the quality and safety of milk powders will be worth doing in the future.

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture-Dependent and DGGE Approaches.

UNLABELLED: Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture-dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture-dependent analysis, the populations of yeasts and LAB were at the level of 10(5) to 10(7) and 10(6) to 10(7) cfu/g, respectively. Similarly, the results of RT-qPCR showed that the values of total yeasts and LAB populations were in the range of 10(6) to 10(7) and 10(7) to 10(8) copies/g, respectively. Using culture-dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR-DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species. PRACTICAL APPLICATION: In this study, microbiota of Chinese sourdoughs was investigated using culture-dependent and PCR-DGGE methods. It will provide deeper insights into the complex microbial ecosystems of sourdough. On the other hand, it will provide opportunity to utilize the potential of selected strains yeasts and LAB to obtain more desirable fermented food.