Production in food of 1,3-pentadiene and styrene by Trichoderma species

The ability of two strains of Trichoderma, isolated from food, to produce the Volatile Organic Compounds 1,3-pentadiene and styrene was investigated. One of the strains had been implicated in a case of food spoilage involving the production of both compounds. In vitro in potato dextrose broth, the strains produced both 1,3-pentadiene and styrene within 5 days in the presence of sorbic acid and cinnamic acid. The taints were produced only in the presence of sorbic acid and cinnamic acid and were not synthesised de novo under the test conditions. Neither the conversion of cinnamic acid to styrene, nor the conversion of sorbic acid to 1,3-pentadiene by Trichoderma strains in foods has been previously reported. The range of organisms implicated in these types of spoilage is thus extended.     

Moniliella acetoabutans: some further characteristics and industrial significance

Summary. The distribution of Moniliella acetoabutans and its behaviour as a spoilage agent of acetic acid preserves, is discussed. the ability of this fungus to tolerate and utilize acetic acid as a nutrient source places it in a unique category. Its characteristics according to the yeast classification of Lodder and Kreger-van Rij are given. the levels of sulphur dioxide, methyl and propyl para -hydroxybenzoates, and sorbic acid which will inhibit this mould at pH 3.3, in the presence of 1.0% acetic acid, have been determined. Sulphur dioxide at 100 ppm, the maximum legal limit, inhibits the mould but its value in practice would be doubtful because of its tendency to combine with aldehydes and so become microbially inactive. Sorbic acid offers a means of controlling spoilage by this mould, were it to be legally permitted.     

High Pdr12 levels in spoilage yeast (Saccharomyces cerevisiae) correlate directly with sorbic acid levels in the culture medium but are not sufficient to provide cells with acquired resistance to the food preservative

Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understanding its effect on the molecular physiology of yeast cells will allow the food industry to develop knowledge-based strategies to make more optimal use of its preservative action. Here we show that the yeast membrane protein Pdr12, previously shown to be prominently involved in sorbic acid resistance development in laboratory strains, was strongly induced by the presence of sorbic acid in the culture medium in Saccharomyces strains isolated from spoiled foods. Induction of Pdr12 expression was seen both under laboratory conditions and upon growth in a commercial soft drink. Induction was rapid and maintained for the duration of the stress. No Pdr12-like protein induction was seen in Zygosaccharomyces bailii or Zygosaccharomyces lentus, two well-known beverages spoilage organisms. Finally, unexpectedly, our studies showed for the first time that pre-inducing Pdr12p to maximal levels by subjecting cells to a mild sorbic acid stress did not lead to cells with an acquired resistance. Neither more rapid growth in the presence of the acid nor growth at higher sorbic acid concentrations at a given environmental pH was observed. Thus we have shown that while important in resistance development against sorbic acid, by itself induction of the pump is not sufficient to acquire resistance to the preservative.     

Fungal metabolites of sorbic acid

A number of fungal detoxification reactions of sorbic acid have been reviewed. These include decarboxylation to give trans-1,3-pentadiene, esterification to give ethyl sorbate, reduction to give 4-hexenol and 4-hexenoic acid. It was shown that seven Penicillium species could convert sorbic acid into 1,3-pentadiene whilst P. bilaii, P. fellutanum and P. glabrum did not. However, most Eurotium species were unable to bring about this conversion. Considerable differences in the resistance of two isolates of P. crustosum to sorbic acid were found. An isolate from coconut was more resistant than one isolated from hazelnuts. Both sorbic acid and caproic acid (hexanoic) brought about disorganization of the mitochondrial membranes in P. crustosum. It is suggested that these lipophilic acids inhibit growth by interfering with the electrochemical membrane potential across the mitochondrial membranes.     

Sorbic acid resistance: the inoculum effect

Zygosaccharomyces is a genus associated with the more extreme spoilage yeasts. Zygosaccharomyces spoilage yeasts are osmotolerant, fructophiles (preferring fructose), highly-fermentative and extremely preservative-resistant. Zygosaccharomyces bailii can grow in the presence of commonly-used food preservatives, benzoic, acetic or sorbic acids, at concentrations far higher than are legally permitted or organolepically acceptable in foods. An inoculum effect has been described for many micro-organisms and antimicrobial agents. The minimum inhibitory concentration (MIC) increases with the size of the inoculum; large inocula at high cell density therefore require considerably higher concentrations of inhibitors to prevent growth than do dilute cell suspensions. A substantial inoculum effect was found using sorbic acid against the spoilage yeast Zygosaccharomyces bailii NCYC 1766. The inoculum effect was not caused by yeasts metabolizing or adsorbing sorbic acid, thereby lowering the effective concentration; was not due to absence of cell-cell signals in dilute cell suspensions; and was not an artefact, generated by insufficient time for small inocula to grow. The inoculum effect appeared to be caused by diversity in the populations of yeast cells, with higher probability of sorbic acid-resistant cells being present in large inocula. It was found that individual cells of Zygosaccharomyces bailii populations, grown as single cells in microtitre plate wells, were very diverse, varying enormously in resistance to sorbic acid. 26S ribosomal DNA sequencing did not detect differences between the small fraction of resistant 'super cells' and the average population. Re-inoculation of the 'super cells' after overnight growth on YEPD showed a normal distribution of resistance to sorbic acid, similar to that of the original population. The resistance phenotype was therefore not heritable and not caused by a genetically distinct subpopulation. It was concluded that resistance of the spoilage yeast Zygosaccharomyces bailii to sorbic acid was due to the presence of small numbers of phenotypically resistant cells in the population.     

Modelling the effects of (green) antifungals, droplet size distribution and temperature on mould outgrowth in water-in-oil emulsions

Prevention of fungal spoilage is a key microbiological issue for the shelf life of fat spreads. Our aim was to assess and model the scope of (natural) antimicrobials for extending shelf life of spreads (water-in-oil emulsions). Production conditions were established to make 60% model fat spreads with reproducible droplet size distributions. The mould vulnerabilities ranged from 1 to 20 weeks. The system allowed feasibility testing of lytic enzymes (Novozym 234) and LMW compounds against Penicillium roqueforti, a key-spoilage mould. The action of Novozym 234, carvacrol, undecanol and dihydrocarveol was benchmarked against sorbate and preservative-free controls under ambient and chilled conditions. Novozym 234 was ineffective to prevent outgrowth of P. roqueforti. Carvacrol, undecanol and dihydrocarveol had limited effects on shelf-life extension compared to sorbate. Fungal growth boundaries of (un-)preserved spreads were modelled. The emulsion droplet size distribution (DSD) was first captured in a mechanistic parameter DSD-I (I = Influence). DSD-I was a move away from the mean droplet diameter D3,3 as sole quantitative droplet-size distribution parameter for mould susceptibility of emulsions. DSD-I is a combination of available water droplets and surface area to initiate and sustain fungal outgrowth. Followup experiments showed that modelling D3,3 and distribution width (e(sigma)) instead of DSD-I gave better results for emulsions with high e(sigma). Empirical predictive models were subsequently developed for the effects of D3,3, e(sigma) and undissociated sorbic acid (HSO) on the shelf life of emulsions.     

Enzymatic formation of styrene during wheat beer fermentation is dependent on pitching rate and cinnamic acid content

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reaction processes simultaneously to the decarboxylation of ferulic‐ and p‐cumaric acid to clove‐like 4‐vinylguaiacol and phenolic 4‐vinylphenol by the same PAD1 and FDC1 decarboxylase enzymes. However, the formation of styrene occurs much faster within the first hours of fermentation. In addition, the conversion of cinnamic acid starts immediately after pitching without an adaption of yeast on the new medium. Only after 120 min does the level of transposition decrease. Moreover, high cinnamic acid content in pitching wort, in combination with an open fermentation management, causes faster and higher styrene formation during this period. In contrast to the formation of 4‐vinylguaiacol, a correlation between pitching rate and styrene formation during open fermentation could be shown. The resulting time interval between styrene and 4‐vinylguaiacol formation provides scope for minimization strategies for styrene, while maintaining the typical wheat beer flavours. Copyright © 2012 The Institute of Brewing & Distilling     

Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy

Lignocellulose was converted into reducing sugars by using saccharification enzymes from cocultivated Trichoderma reesei and Aspergillus niger and reducing sugars as nutrients for Zymomonas mobilis to produce bioethanol in an immobilization system. After 96 h of cultivation, cocultivated T. reesei and A. niger had enzymatical synergistic effects that enabled a reducing sugar production of 1.29 g/L and a cellulose conversion rate of 23.27%. An 18% total inoculum concentration and a 1/1 inoculation ratio of T. reesei to A. niger obtained a reducing sugar production rate and a cellulose conversion rate of 2.57 g/L and 46.27%, respectively. The co-immobilization cultivation results showed that using polyurethane as a carrier optimized total saccharification enzyme activity at an inoculum ratio of 1/1 and a total inoculum concentration of 6.5×10(6)spores/mL. Based on the experimental results, the bioreactor design was further modified to enhance bioethanol production. The three strains (A. niger, T. reesei and Z. mobilis) were cocultivated with a co-immobilization cultivation system. The experimental results showed that, after 24 h cultivation, bioethanol production reached 0.56 g/L, and reducing sugar conversion rate reached 11.2% when using carboxymethylcellulose (CMC) substrates. The experimental results confirmed that the modified bioreactor enhances bioethanol production. However, further experiments are needed to determine how to prevent multi-stage failure of reducing medium volume.     

Heat Resistance of Eurotium herbariorum, a Xerophilic Mold

Eurotium haerbariorum, isolated from a spoilage outbreak involving grape preserves, was a true osmophile in that optimal growth was obtained in media containing 40-60% sucrose. Survival curves showed logarithmic death of heated spores followed by a tailing. In 5° Brix grape juice D-values at 70°C and z-values were 2.5 min and 9.1°C compared to 5.2 min and 7.1°C in 65°C Brix juice. Low concentrations of sorbic acid and fumaric acid in the heating menstruum had little effect on heat resistance.     

Efficient synthesis of hydroxystyrenes via biocatalytic decarboxylation/deacetylation of substituted cinnamic acids by newly isolated Pantoea agglomerans strains

BACKGROUND: Decarboxylation of substituted cinnamic acids is a predominantly followed pathway for obtaining hydroxystyrenes—one of the most extensively explored bioactive compounds in the food and flavor industry (e.g. FEMA GRAS approved 4‐vinylguaiacol). For this, mild and green strategies providing good yields with high product selectivity are needed. RESULTS: Two newly isolated bacterial strains, i.e. Pantoea agglomerans KJLPB4 and P. agglomerans KJPB2, are reported for mild and effective decarboxylation of substituted cinnamic acids into corresponding hydroxystyrenes. Key operational parameters for the process, such as incubation temperature, incubation time, substrate concentration and effect of co‐solvent, were optimized using ferulic acid as a model substrate. With strain KJLPB4, 1.51 g L^?1 4‐vinyl guaiacol (98% yield) was selectively obtained from 2 g L^?1 ferulic acid at 28 °C after 48 h incubation. However, KJPB2 provided vanillic acid in 85% yield after 72 h following the oxidative decarboxylation pathway. In addition, KJLPB4 was effectively exploited for the deacetylation of acetylated α‐phenylcinnamic acids, providing corresponding compounds in 65–95% yields. CONCLUSION: Two newly isolated microbial strains are reported for the mild and selective decarboxylation of substituted cinnamic acids into hydroxystyrenes. Preparative‐scale synthesis of vinyl guaiacol and utilization of renewable feedstock (ferulic acid extracted from maize bran) have been demonstrated to enhance the practical utility of the process. Copyright © 2011 Society of Chemical Industry     

三华李果坯发酵液中腐败真菌分离鉴定、相关抑菌剂效价评定及发酵工艺优化

为了探究三华李果坯接种乳酸菌发酵异常的原因,改善发酵工艺,以表面长有白色菌膜的异常发酵液为分离源,对其中的腐败真菌进行分离纯化,通过形态学特征观察和ITS序列鉴定,得到3株优势腐败真菌,分别为Candida stellimalicola、Pichia kudriavzevii和Pichia occidentalis;用牛津杯法评定了山梨酸、纳他霉素及二甲基二碳酸盐的抑菌效价,发现3种抑菌剂作用于上述3株腐败真菌,均出现了明显抑菌圈,二甲基二碳酸盐对发酵所用的干酪乳杆菌略有抑制作用,抑菌圈直径为（10.45 ± 0.11）mm,说明3种抑菌剂可以用于抑制腐败真菌的生长繁殖但不会影响发酵的正常进行;用棋盘稀释法测定了抑菌剂的联合抑菌效力,山梨酸与纳他霉素联合作用于Pichia kudriavzevii的部分抑菌浓度指数为0.50;山梨酸与二甲基二碳酸盐联合作用于Candida stellimalicola和Pichia occidentalis的部分抑菌浓度指数分别为0.50和0.38,均表现出协同抑菌作用;采用响应面法优化发酵防腐工艺,显示在发酵液中按果重添加质量浓度0.040%山梨酸、0.010%纳他霉素和0.015%二甲基二碳酸盐,室温密封发酵30 d后,测得发酵液中霉菌酵母活菌数为3.212 lg CFU/mL,远低于旧工艺发酵液中霉菌酵母活菌数,发酵液表面不再出现白膜。新工艺简单有效、效果稳定,解决了发酵异常导致的果坯软化、产生异味等问题。     

Inhibition of Bacillus subtilis and Listeria innocua by nisin in combination with some naturally occurring organic compounds

The application of combined preservative factors (hurdle technology) is very effective in controlling the growth of food spoilage and foodborne pathogenic bacteria. Antimicrobial activity of nisin alone and in combination with some natural organic compounds (carvacrol, cinnamic acid, eugenol, diacetyl, and thymol) on the growth of gram-positive bacteria Bacillus subtilis and Listeria innocua was-investigated. All the organic compounds tested exhibited antimicrobial activity against the microorganisms used; however, the MICs varied between 0.8 and 15.0 mM depending on the potency of the compound or the sensitivity of the target strain. Investigation of the interaction between the organic compounds and nisin against the test organisms revealed different patterns, varying from synergistic to antagonistic. Combinations of nisin with carvacrol, eugenol, or thymol resulted in synergistic action against both test organisms. Activity of nisin and cinnamic acid together was synergistic against L. innocua, but only additive against B. subtilis. In contrast, the combination of diacetyl and nisin resulted in an antagonistic effect against both test organisms. This study highlights the potential of the combination of these compounds with nisin to inhibit pathogen growth in food.     

Formation of styrene dependent on fermentation management during wheat beer production

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reactions proceed in parallel to the decarboxylation of ferulic- and p-cumaric acid to 4-vinylguaiacol and 4-vinylphenol by the same decarboxylase enzyme. However, the formation of styrene occurs much faster and all available cinnamic acid in wort was converted completely within a few hours. Moreover, the comparison of various manufacturing parameters shows that a higher fermentation temperature of 25 °C compared to 16 °C and an open fermentation management lead to a rapid decrease of styrene. This allows minimising the content of styrene in beer while maintaining the typical wheat beer flavours.     

Physiological characterization of spoilage strains of Zygosaccharomyces bailii and Zygosaccharomyces rouxii isolated from high sugar environments

Two isolates of spoilage yeasts Zygosaccharomyces bailii and Zygosaccharomyces rouxii were obtained from a high sugar environment, in a factory producing candied fruit and nougat. Other strains, Z. bailii from other environments and other isolates from high sugar/salt environments were obtained for comparison (Zygosaccharomyces lentus, Candida magnoliae, Candida halophila and Pichia guilliermondii). A full physiological assessment of these isolates was carried out, of preservative and biocide resistance, osmotolerance, ethanol-tolerance, low pH resistance, degree of fermentation, and growth temperature and survival to pasteurisation. Results showed that the strains isolated from high sugar environments did not show extreme physiology. These were robust strains but within the normal parameters expected for the species. One exception to this was that the Z. bailii strains were abnormally able to grow at 37 degrees C. In all strains other than C. magnolia and C. halophila, cells were able to adapt to high levels of sugar. Cultures grown in high glucose concentrations were subsequently able to tolerate higher concentrations of glucose than previously. Similarly, high sugar was found to confer a degree of protection against pasteurisation, enabling survival in what would have otherwise been a lethal treatment. Isolates of Z. bailii showed a high level of resistance to preservatives such as sorbic acid, benzoic acid, acetic acid, cinnamic acid, and ethanol, and also to heat. Unexpectedly Z. bailii isolates were not exceptionally resistant to biocides such as peracetic acid, or hypochlorite. These results indicate that spoilage by yeasts such as Z. bailii may be better prevented by use of biocidal cleaning agents in the factory, rather than treating the food with preservatives.     

Antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris vegetative cells in apple juice

The aim of this study was to investigate the antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris. Minimum inhibitory concentrations of cinnamic acid and chlorogenic acid were 0.375 and 2.0 mg mL⁻¹, and the minimum bactericidal concentrations were 0.50 and 4.0 mg mL⁻¹, respectively. The apple juice ingredients had little influence on the inactivation of A. acidoterrestris. After treatment with cinnamic acid and chlorogenic acid, the morphology of A. acidoterrestris cells were severely destroyed; the leakage of nucleic acids and proteins increased significantly. SDS-PAGE investigation of bacterial proteins proved that the loss of soluble proteins was obvious as well. These results demonstrated that cinnamic acid and chlorogenic acid exerted their antibacterial activity mainly by an action mode of membrane disruption. This study provides an alternative method for the control of A. acidoterrestris-related spoilage in the fruit juice/beverage industry.     

Metabolism of phenylalanine and biosynthesis of styrene in Penicillium camemberti

The occurrence of styrene in food may be an important aroma defect (celluloid odour), even at very low concentrations (Miltz et al. 1980) causing consumer rejection and is therefore a problem for the food industry. We examined the biosynthetic pathway leading to styrene formation by Penicillium camemberti using labelled compounds. As styrene is strongly hydrophobic and volatile, we first had to develop a continuous extraction process. Using resins XAD2 it was reasonable to suspect phenylalanine (Phe) as the precursor. The addition of Phe marked with 13C on the ring provokes the accumulation of labelled styrene. The enzyme activities involved were also tentatively measured. Styrene appears to be synthesized from phenylalanine by phenylalanine ammonia-lyase activity followed by a decarboxylation catalysed by a cinnamic acid decarboxylase.     

Transformation of substituted cinnamic acids using l-cysteine metal complexes in aqueous media

4-Hydroxycinnamic, 4-methoxycinnamic, ferulic and cinnamic acids were both non-oxidatively and oxidatively decarboxylated in alkaline aqueous media in the presence of l-cysteine–Fe(II) and l-cysteine–Co(II) heterogeneous catalysts using hydrogen peroxide or molecular oxygen. GC/MS analysis of diethylether extracts of reaction mixtures confirmed that the addition of hydrogen peroxide resulted predominantly in oxidative decarboxylation of substituted cinnamic acids, producing the corresponding carbonyl compounds (4-hydroxybenzaldehyde, 4-methoxybenzaldehyde, vanillin, benzaldehyde). On the other hand, saturation of this heterogeneous reaction system with molecular oxygen led to the formation of a variety of products, probably via peroxoacid anions or peroxoradical intermediates, e.g., ferulic acid was transformed to vinylguaiacol and vanillin with yields of 22% and 0.7%, respectively.     

牛肉低温贮藏环境中沙门氏菌诱导耐酸响应的存在程度及其产生机制

研究牛肉生产和销售过程中沙门氏菌诱导耐酸响应（acid tolerance response,ATR）的存在程度及其产生机制。探究微酸诱导、双组分基因敲除、低温长期贮藏对沙门氏菌耐酸能力的影响,同时借助λRed同源重组、实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）及氨基酸添加实验探索菌株耐酸性产生的内在机理。结果表明,微酸诱导能够显著增强沙门氏菌的耐酸能力（P＜0.05）,并且ATR一旦形成,在牛肉低温贮藏（4 ℃）的过程中至少可以维持7 d,对食品安全有极大危害。牛肉培养基作为微酸的细菌生长介质,在低温下其本身并不能引发沙门氏菌产生ATR,说明低温处理可能是抑制沙门氏菌ATR的重要方法。real-time PCR和氨基酸添加实验表明,沙门氏菌的双组分系统PhoP/PhoQ和PmrA/PmrB均参与酸性环境的感知,并能通过调控精氨酸脱羧和赖氨酸脱羧系统提高菌株的耐酸性,这从氨基酸代谢角度解释了沙门氏菌诱导ATR的产生机制,同时也揭示了食品基质提升微生物耐酸性这一表观现象的内在机理。     

Effect of Flavonoids and Related Compounds on Soybean Lipoxygenase-1 Activity

Soybean lipoxygenase-1 activity was determined in the presence of the flavonoid compounds quercetin, rutin and kaempferol, and with caffeic acid and chlorogenic acid, which are cinnamic acids. All flavonoids and related compounds resulted in at least 10% inhibition of lipoxygenase activity on an equal weight basis. Inhibition of lipoxygenase-1 activity by flavonoids and cinnamic acids was compared to inhibition by butylated hydroxyanisole (BHA) and butylated hydrox-ytoluene (BHT). Kaempferol, caffeic acid and chlorogenic acid showed inhibition comparable to that of BHT. On an equimolar basis, rutin was the most effective inhibitor of lipoxygenase activity.