Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.     

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

细菌群体感应及其在食品变质中的作用

食品相关细菌引起的生物被膜形成和食品变质是食品工业中的重大问题。研究表明细菌群体感应(Quorum sensing,QS)与被膜形成、食品腐败变质密切相关。重点对细菌产生的各种QS信号分子及其在被膜形成的作用和被膜在食品工业中的重要性做了介绍。QS信号分子与食品变质密切相关,故对QS抑制剂作为新型食品防腐剂以延长储存期限及加强食品安全的前景进行了概述。     

Genome sequence of a food spoilage lactic acid bacterium, Leuconostoc gasicomitatum LMG 18811T, in association with specific spoilage reactions

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modified-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas and slime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats have especially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sausages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG 18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to study its phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was done within L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG 18811T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%. It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. As interesting features associated with the growth and spoilage potential, LMG 18811T possesses utilization strategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electron transport chain requiring only externally supplied heme for respiration. In respect of the documented specific spoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formation were recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, but their functionality and role in food spoilage and processing environment contamination need further study.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.     

Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid‐tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN‐ARS‐based CRISPR‐Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site‐specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR‐Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.     

3,4‐dimethoxystilbene,a resveratrol derivative with anti‐angiogenic effect,induces both macroautophagy and apoptosis in endothelial cells

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013. © 2012 Wiley Periodicals, Inc.     

The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not only YME2 functions in Saccharomyces cerevisiae, but also the capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives

A factor influencing resistances of food spoilage microbes to sorbate and benzoate is whether these organisms are able to catalyse the degradation of these preservative compounds. Several fungi metabolize benzoic acid by the beta-ketoadipate pathway, involving the hydroxylation of benzoate to 4-hydroxybenzoate. Saccharomyces cerevisiae is unable to use benzoate as a sole carbon source, apparently through the lack of benzoate-4-hydroxylase activity. However a single gene from the food spoilage yeast Zygosaccharomyces bailii, heterologously expressed in S. cerevisiae cells, can enable growth of the latter on benzoate, sorbate and phenylalanine. Although this ZbYME2 gene is essential for benzoate utilization by Z. bailii, its ZbYme2p product has little homology to other fungal benzoate-4-hydroxylases studied to date, all of which appear to be microsomal cytochrome P450s. Instead, ZbYme2p has strong similarity to the matrix domain of the S. cerevisiae mitochondrial protein Yme2p/Rna12p/Prp12p and, when expressed as a functional fusion to green fluorescent protein in S. cerevisiae growing on benzoate, is largely localized to mitochondria. The phenotypes associated with loss of the native Yme2p from S. cerevisiae, mostly apparent in yme1,yme2 cells, may relate to increased detrimental effects of endogenous oxidative stress. Heterologous expression of ZbYME2 complements these phenotypes, yet it also confers a potential for weak acid preservative catabolism that the native S. cerevisiae Yme2p is unable to provide. Benzoate utilization by S. cerevisiae expressing ZbYME2 requires a functional mitochondrial respiratory chain, but not the native Yme1p and Yme2p of the mitochondrion.     

The effect of potassium sorbate, NaCl and pH on the growth of food spoilage fungi

In this study, the hurdle technology approach was used to prevent fungal growth of common spoilage fungi in naturally fermented black olives (Alternaria alternata, Aspergillus niger, Fusarium semitectum andPenicillium roqueforti). The factors studied included a combination of different concentrations of potassium sorbate (100 up to 1000 mg/L), a range of pH values (4.5, 5, 5.5, 6, and 6.5) and levels of NaCl (0, 3.5, 5, 7.5, and 10%).Alternaria alternata was the most sensitive fungus whereasP. roqueforti was the most resistant fungi against all hurdle factors. The combination of all hurdles completely inhibitedA. alternata andF. semitectum by lowest inhibitory factors, such as 100 mg/L potassium sorbate with 3.5% NaCl at pH 5. On the other hand, at pH 5, A.niger andP. roqueforti were totally prevented by a combination of 300 mg/L potassium sorbate with 10% NaCl and 400 mg/L potassium sorbate with 7.5% NaCl, respectively. Potassium sorbate and 5–10% NaCl interaction had significant stimulation effect onp. roqueforti andA. niger (p<0.05). This study indicates that potassium sorbate is a suitable preserving agent to inhibit growth of fungi in fermented products of pH near 4.5 regardless levels of NaCl. For products of slightly higher pH, the addition of potassium sorbate is suggested in combination with NaCl.     

Immunization with a combination of recombinant Brucella abortus proteins induces T helper immune response and confers protection against wild-type challenge in BALB/c mice

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis.     

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death.     

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2)

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.     

Degradation of phenol and benzoic acid in a three-phase fluidized-bed reactor

Degradation of phenol and benzoic acid was studied in a fluidized-bed reactor (liquid volume 2.17 L) under nonsterile conditions with special emphasis on maximizing the flow through the reactor and investigating reactor performance at fluctuating feeds. Reactor response to substrate pulses was investigated by applying substrate square-wave inputs at a liquid flow of 1.00 L h(-1). A twofold increase of the phenol and benzoic acid feed concentrations for 2.5 h did not lead to accumulation and breakthrough. The cells were able to survive four to fivefold increases of the feed concentration for 1 h without loss of viability, although the phenol pulse lead to phenol accumulation in the reactor. Reactor performance at constantly fluctuating loads was investigated by varying the feed concentrations using sine wave functions. No accumulation of phenol or benzoic acid was observed. Influence of induction was studied using shift experiments. After 35 days of operation (369 hydrodynamic residence times) with phenol as sole substrate (carbon source) the reactor was able to mineralize benzoic acid without any adaptation or lag phase. The capability of phenol degradation, on the other hand, was lost by most cells after only 3 days operation with benzoic acid as the sole substrate. The experiments underline the importance of induction. In order to maximize the flow through the reactor, the liquid flow was increased stepwise while the feed concentrations were reduced correspondingly, keeping the volumetric conversion rates of phenol (0.24 g L(-1) h(-1)) and benzoic acid (0.17 g L(-1) h(-1)) constant. By this means, liquid flow could be increased up to 13.32 L h(-1), which was more than 20-fold higher than the maximum liquid flow achievable in a chemostat using the same conditions.     

Inhibitory effect of roburic acid in combination with docetaxel on human prostate cancer cells

Roburic acid (ROB) is a naturally occurred tetracyclic triterpenoid, and the anticancer activity of this compound has not been reported. Docetaxel (DOC) is the first-line chemotherapeutic agent for advanced stage prostate cancer but toxic side effects and drug resistance limit its clinical success. In this study, the potential synergistic anticancer effect and the underlying mechanisms of ROB in combination with DOC on prostate cancer were investigated. The results showed that ROB and DOC in combination synergistically inhibited the growth of prostate cancer cells. The combination also strongly induced apoptosis, and suppressed cell migration, invasion and sphere formation. Mechanistic study showed that the combined effects of ROB and DOC on prostate cancer cells were associated with inhibition of NF-κB activation, down regulation of Bcl-2 and up regulation of Bax. Knockdown of NF-κB by small interfering RNA (siRNA) significantly decreased the combined effect of ROB and DOC. Moreover, we found that esomeprazole (ESOM), a proton pump inhibitor (PPI), strongly enhanced the effectiveness of ROB and DOC on prostate cancer cells in acidic culture medium. Since acidic micro environment is known to impair the efficacy of current anticancer therapies, ESOM combined with ROB and DOC may be an effective approach for improving the treatment of prostate cancer patients.     

Modelling the effect of temperature and water activity on growth of Aspergillus niger strains and applications for food spoilage moulds

AIMS: To develop a model for the combined effect of water activity (a(w)) and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature. METHODS AND RESULTS: An extended combined model describing the growth of two strains of A. niger, as a function of temperature (25-30 degrees C) and a(w) (0.90-0.99) was developed. The growth rate (micro) was expressed as the increase in colony radial growth per unit of time. This extends the previous square root model showing the relationship between temperature and bacterial growth rate developed by Ratkowsky et al. (1983) and the parabolic relationship between the logarithm of the growth rate and a(w) developed by Gibson et al. (1994). A good correlation between the experimental data and the model predictions was obtained, with regression coefficients (r(2)) > 0.99. In addition, the use of this model allowed predictions of the cardinal a(w) levels: a(w(min)), and a(w(opt)). The estimation of the minimum a(w) levels (a(w(min))) was in accordance with data in the literature for similar and a range of other Aspergillus and related species, regardless of the solutes used for a(w) modification. The estimation of the optimal a(w) (a(w(opt))) and the optimal growth rate (micro(opt)) were in good agreement with the experimental results and data from the literature. CONCLUSIONS: This approach enables accurate prediction of the combined effects of environmental factors on growth of spoilage fungi for rapid prediction of cardinal limits using surface response curves. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a rapid method for predicting optimal and marginal conditions for growth of a wide range of spoilage micro-organisms in relation to interacting environmental conditions and will have applications for improving shelf-life of intermediate moisture foods.     

Caffeine exerts a dual effect on capacitative calcium entry in Xenopus oocytes

Caffeine increases the amplitude of the Cl- currents evoked by capacitative Ca2+ entry (CCE) on thapsigargin-treated Xenopus oocytes. The caffeine-induced potentiation of the CCE process appears to rest on two distinct and additive components. The first component involves the cAMP second messenger system since it can be mimicked by either IBMX perfusion or cAMP microinjection into the oocyte and inhibited by the PKA inhibitory peptide i-PKA. The second component, although activatory, is dynamically related to the caffeine-evoked inhibition of InsP3-mediated Ca+ release and may arise from an interaction between caffeine and the InsP3 receptor in the context of a conformational coupling between the InsP, receptor and the channels responsible for CCE.     

The effect of benzoic acid concentration on nitrogen metabolism,manure ammonia and odour emissions in finishing pigs

A complete randomised block design experiment was conducted to investigate the effects of benzoic acid inclusion level on nitrogen (N) metabolism, and manure ammonia (NH₃) and odour emissions in finishing pigs. Sixteen boars (64 kg live weight ± 1.5 kg) were assigned to one of four dietary treatments (T) varying in benzoic acid concentration: (T1) 0 g benzoic acid/kg (as fed); (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; (T4) 30 g benzoic acid/kg. Animals were housed in individual metabolism crates and feed was provided ad libitum. All diets were formulated to have similar concentration of digestible energy and ileal digestible lysine with benzoic acid replacing wheat in the diet. There was a linear decrease in NH₃ emission (P<0.001), as the dietary benzoic acid concentration increased (141.4 mg/g versus 40.5 mg/g N intake (S.E.M. 12.1) over the 240-h storage period). However, there was no effect (P>0.05) of benzoic acid on odour concentration. Urinary nitrogen (N) excretion, total N excretion and the urinary:faecal N ratio were linearly reduced (P<0.05) with increasing benzoic acid inclusion. Furthermore, N retention increased linearly (P<0.05) as benzoic acid concentration increased from 0 g/kg to 30 g/kg in the diet. In conclusion, the inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary N excretion and the urinary to faecal N ratio. This was mirrored by reductions in manure NH₃ emissions in the benzoic acid supplemented treatments.