Determination of 4-hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) in cultures of bacteria

4-Hydroxy-2,5-dimethyl-3(2#)-furanone (HDMF) was detected at concentrations of 598 μg/l and 427 μg/l, respectively, after 7 days of growth ofLactobacillus helveticus andL. delbrueckii on an aqueous suspension of whey powder. In contrast, a culture ofPropionibacterium freudenreichii contained only 83 μg/l HDMF. The results suggest that lactic acid bacteria cause the production of HDMF in Swiss cheese.     

产4-羟基-2,5-二甲基-3(2H)-呋喃酮酵母菌的分离、筛选及分子生物学鉴定

以菠萝、草莓等水果为分离源,分离、筛选利用D-果糖产香料HDMF酵母菌株,并对其高产菌株进行分子生物学鉴定。采用稀释涂布法分离酵母菌株,高效液相色谱法检测HDMF产量,26SrDNAD1/D2区序列分析及系统发育分析鉴定菌株。共分离得到46株酵母,5株可利用D-果糖产HDMF;产量较高的两株菌为:C5(6.84mg/L)、P3(10.96mg/L),分别约为已报道毕赤氏酵母属(Pichia capsulata)利用L-(+)-鼠李糖产HDMF的3倍和5倍;26S rDNA D1/D2区序列分析及系统发育分析结果显示,P3与Pichia caribbica(毕赤酵母属),C5与Hanseniaspora sp.(有孢汉逊酵母属)相似性均在99%以上,分子生物学法鉴定P3为Pichia caribbica,C5为Hanseniaspora sp.     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH 2.0–8.0, interval: 1.5 pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23 °C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; R?hm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved. Received: 25 September 1996     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH?2.0–8.0, interval: 1.5?pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23?°C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; Röhm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved.     

高产2,5-二甲基-4-羟基-3(2H)-呋喃酮(DMHF)酵母菌株的选育

从酒曲中筛选到1株高产2,5-二甲基-4-羟基-3(2H)-呋喃酮(DMHF)的酵母菌株(SX-21).高浓度NaCl和外源添加1,6-二磷酸果糖有利SX-21菌合成DMHF,其产量为100mg/L,达到文献报道的最高产量.经NTG诱变,筛选到1株突变株(NTG-SX-103),其产量提高到320 mg/L.SX-21菌株经18S rRNA鉴定为季也蒙毕氏酵母.     

L-Rhamnose: progenitor of 2,5-dimethyl-4-hydroxy-3 [2H]-furanone formation by Pichia capsulata?

 2,5-Dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, 1), an important aroma constituent, was detected at concentrations of up to 2 mg/l after 4 days of growth of Pichia capsulata on casein peptone culture medium containing L-(+)-rhamnose (2). Blank samples without yeast contained no 1 after the incubation period. In parallel experiments five samples of 2 exhibiting different [¹³C] abundance were given to P. capsulata. The volatile compounds formed were isolated and analysed for their [¹³C]/[¹²C] ratios by on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). A positive correlation between the isotopes in 1 and 2 was observed; thus, 2 served as the carbon source for 1. However, 1 was formed from a postulated intermediate of 2 generated during thermal sterilization, as 1 was neither detected after sterile filtration of the culture medium nor after separate thermal sterilization of the casein peptone and 2. This observation was confirmed by an experiment investigating the time course of the formation of 1. Received: 4 June 1996     

微生物发酵法制备4-羟基-2,5-二甲基-3[2H]-呋喃酮的研究进展

4-羟基-2,5-二甲基-3[2H]-呋喃酮(HDMF)是一种很好的食品调味剂,具有很高的商业价值.文中分析了微生物发酵法制备HDMF的优越性,总结了产生HDMF的菌种,论述了对其产生机理进行的探索,说明了目前存在的问题,并展望了运用现代生物技术手段改良菌种、构建幕因工程菌株、达到高产HDMF的研究前景.     

Uncoupling of growth and exopolysaccharide production by Lactococcus lactis subsp. cremoris NIZO B40 and optimization of its synthesis

Exopolysaccharide (EPS) production by Lactococcus lactis subsp. cremoris NIZO B40 was found to be most efficient with glucose as a substrate. The optimal temperature and pH for EPS synthesis were 25 degrees C and pH 5.8, respectively. EPS production could not be identified as a stress response: increased oxygen tension and reduced water activity negatively affected both growth and EPS synthesis. It is often assumed that there is a competition between growth and EPS formation. Within the range of 0.5 and 0.1 h(-1), reducing the growth rate resulted indeed in an increase of the specific EPS production but the polymer formation decreased again at even lower growth rates. Most of the applied fermentation conditions influenced both growth and EPS formation. As the growth rate itself also influenced EPS formation, we studied the linking between growth and EPS synthesis. Interestingly, EPS production was not strictly coupled to growth. Significant de novo synthesis of EPS was observed in non-growing cultures. Consequently, the influence of different culture conditions on EPS production could be studied independent of growth.     

Estimation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (FURANEOLA®) in cultivated and wild strawberries,pineapples and mangoes

The amount of FURANEOLA® in strawberries, pineapples, and mangoes has been determined by quantitative gas chromatography after ethyl ether extraction of the fruits.     

Amino acid-dependent formation pathways of 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone in the Maillard reaction

The formation pathways of two furanoids, 2-acetylfuran and 2,5-dimethyl-4-hydroxy-3[2H]-furanone (DMHF) were studied by GC–MS in the Maillard-type model system based on glucose and selected amino acids. The reaction was performed in 0.01 M phosphate buffer by heating a 1:1 mixture of [¹³C6] glucose and [¹²C6] glucose with amino acid. There is only one major formation pathway for DMHF in which the glucose carbon skeleton stayed intact. Formation pathways for 2-acetylfuran were more complicated. They formed either from glucose or from glucose and glycine. In the presence of glycine, the [C-5] unit of glucose combined with formaldehyde from glycine leads to 2-acetylfuran. For other amino acids, either cyclisation of intact glucose or recombination of glucose fragments can lead to 2-acetylfuran formation. These results indicate a competitive trend in controlling Maillard reaction. Therefore, besides changing Miallard reaction impact factors (temperature, time, pH etc.), inhibiting or preventing the competitive reaction cascade may direct desired pathways of Maillard reaction.     

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as a secondary metabolite from D-fructose-1,6-diphosphate metabolism by Zygosaccharomyces rouxii

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of d-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g.l(-1)) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by Z. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g.l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-13C-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of (13)C(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.     

Use of Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris isolated from TRFM in manufacturing of functional low-fat cheeses

Abstract: The purpose of this study was to manufacture new functional low‐fat cheeses using Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris strains isolated from TRFM. After 28 d of ripening and storage, the viable populations of lactic acid bacteria (LAB) in the low‐fat cheeses made with L. lactis subsp. cremoris TL1 (TL1), L. lactis subsp. cremoris TL4 (TL4), and TRFM still maintained above 10⁸ CFU/g. The low‐fat cheeses made with TL1 and TRFM showed higher moisture contents than the cheeses made with TL4, full‐fat, and low‐fat cheese controls. The low‐fat cheeses made with TL1 and TL4 had higher customer preferential scores similar to full‐fat cheese control in the sensory evaluation. Additionally, the low‐fat cheeses fermented with TL1, TL4, and TRFM for 4 h had higher 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical‐scavenging and ferrous ion‐chelating abilities than the cheeses fermented with the starters for 8 h, full‐fat, and low‐fat cheese controls. A better angiotensin‐converting enzyme (ACE) inhibition activity was also observed in the low‐fat cheeses made with TL1, TL4, and TRFM than that in the full‐fat and low‐fat cheese controls during ripening and storage period. Practical Application: As health‐conscious consumers continue to seek low‐fat alternatives in their diets, there remain strong interests for the dairy industry to develop low‐fat cheeses to meet the demands. This study clearly demonstrated that the low‐fat cheeses fermented with TL1 for 4 h showed a better overall acceptability and possessed antioxidative abilities and ACE inhibitory activities than other cheeses tested in this study. By improving its flavor and investigating the possible mechanisms of its functionalities in the future, this low‐fat cheese might possibly be commercialized and give a positive impact on cheese consumption in the future.     

High Performance Liquid Chromatography of 2,5-Dimethyl-4-Hydroxy-3 (2H)-Furanone in Pineapple and Grapefruit Juices

A reversed-phase HPLC method was developed for rapid analysis of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), the compound responsible for the “burnt-pineapple” flavor in pineapple juices. A Zorbax ODS column was used with a mobile phase of 0.05M sodium acetate (pH 4.0)/methanol (70:30); detection was by UV at 290 nm. Sample clean up was accomplished by solid-phase extraction with C-18 Sep-Pak cartridges. DMHF contents ranged from 1.6 to 27.3 ppm from 10 fresh pineapple juices. Using this system, the production of DMHF was monitored in canned grapefruit juices during storage at varying temperatures.     

4-羟基-2,5-二甲基-3(2H)呋喃酮对单增李斯特菌AI-2类群体感应的干扰效应

利用群体感应抑制剂靶向干扰病源菌已成为有效控制致病性危害的突破口。本研究通过测定菌体浓度、哈氏弧菌BB170报告菌的发光值,研究不同浓度4-羟基-2,5-二甲基-3(2H)呋喃酮(DMHF)作用下单增李斯特菌(L.m)的生长与信号分子AI-2活性;通过半固体穿刺法、结晶紫法及溶血平板法检测L.m的动力、生物膜及溶血性,来评价DMHF对L.m AI-2类QS的干扰效应。结果表明,≤ 200 μg/mL的DMHF能推迟L.m的生长,且明显抑制了L.m AI-2的活性,实验组的AI-2活性值均低于阴性对照组的40%,由此可判定DMHF是AI-2类QS的抑制剂;当DMHF浓度为100 μg/mL时能明显抑制L.m的运动能力;50、100和200 μg/mL的DMHF对L.m生物膜的形成抑制率分别为29.72%、44.88%和75.27%;200 μg/mL的DMHF完全抑制溶血环的产生,本研究为利用DMHF作AI-2类QS的抑制剂提供依据。     

Candida etchellsii产2(5)-乙基-4-羟基-5(2)-甲基-3(2H)-呋喃酮的影响因素

在埃切假丝酵母(Candida etchellsii)发酵过程中,通过盐度调控和氨基酸添加,强化目标产物HEMF(2(5)-乙基-4-羟基-5(2)-甲基-3(2H)-呋喃酮)的合成效率。分阶段调控发酵体系的盐度(初始阶段控制CaCl浓度为200 g/L,发酵40 h后提升至220 g/L),结合氨基酸添加(向发酵体系中添加丙氨酸、精氨酸和甘氨酸各1.67 g/L)。摇瓶结果表明:酵母C.etchellsii合成HEMF,其产量为110.74 mg/L。7 L发酵罐上罐验证,HEMF产量达到121.51 mg/L,相比空白(200 g/L CaCl浓度下且没有添加氨基酸)提高了21.2倍。分阶段盐度调控结合氨基酸添加策略显著强化了C.etchellsii对HEMF的合成。     

Biocontrol of Listeria monocytogenes in a model cultured milk (lben) by in situ bacteriocin production from Lactococcus lactis ssp. lactis

Bacteriocin‐producing (Bac⁺) Lactococcus lactis ssp. lactis CCMM/IAV/BK1 isolated from traditional lben was used in the preparation of lben from pasteurized milk to assess its potential inhibitory activity against Listeria monocytogenes ATCC 7644. Production of bacteriocin (arbitrary units, AU) in MRS broth fortified with yeast extract (MRSY) in a fermentor under controlled and uncontrolled pH conditions was also investigated. This Bac⁺ strain yielded about 35 times more bacteriocin when the pH was maintained constant at 6.5 than under varying pH conditions. To test the effect of in situ bacteriocin production against L. monocytogenes, lben was made from cow's milk artificially contaminated with approximately 10⁷ cfu/mL and fermented with a mixed mesophilic starter culture consisting of the lactococcal Bac⁺ organism and Lc. lactis ssp. lactis biovar diacetylactis 66, a diacetyl‐producing strain, in a ratio of 1 : 1. Numbers of L. monocytogenes were monitored during fermentation and storage of lben at refrigeration temperature (c. 7°C) for up to 6 days. Performances of the Bac⁺ starter were compared to those of an isogenic Bac^? derivative strain obtained from the Bac⁺ starter by curing with ethidium bromide. The results showed that the amount of L. monocytogenes decreased to below the detectable level in a 1‐mL sample within 24 h of storage at 7°C in lben fermented with the Bac⁺ starter culture. On the contrary, L. monocytogenes survived for 6 days of storage at 7°C in lben made with the Bac^? starter. The Bac⁺ wild strain of the starter studied could be adequately used to produce lben or similar indigenous fermented milks of improved hygienic quality on an industrial scale. Alternatively, it could be used as an adjunct in minimally processed products or in products obtained from raw milk to add a safety factor.     

Combined pressure–temperature effects on the chemical marker (4-hydroxy-5-methyl- 3(2H)-furanone) formation in whey protein gels

Chemical markers, such as furanone, are intrinsically formed in foods at elevated process temperatures, and have been successfully used as indirect indicators of heating patterns in advanced thermal proces-ses such as aseptic processing, microwave sterilization and ohmic heating. However, very limited information is available on suitability of these chemical markers during combined pressure–heat treatment. The present study was conducted on the formation and stability of chemical marker M-2 (4-hydroxy-5-methyl-3(2H) furanone, a by-product of Maillard reaction) as a function of pressure, temperature and pH. Whey protein gels (containing 1g ribose/100g gel mix) at pH 6.1 and 8.3 were subjected to pressure assisted thermal processing (PATP; 350 and 700 MPa, 105 °C), high pressure processing (HPP; 350 and 700 MPa, 30 °C) and thermal processing (TP; 0.1 MPa, 105 °C) for different holding times. Unprocessed gel was used as control. The marker yield was quantified using HPLC. The initial concentrations of M-2 in the gels were 9.17 and 6.1 mg/100 g at pH 6.1 and 8.3, respectively. As expected, heat treatment at 105 °C, 0.1 MPa increased M-2 concentration. The marker yield increased with increase in holding time, following a first order kinetics and decreased with increasing pH. Pressure treatments from 350 to 700 MPa at 30 °C reduced the chemical marker formation for both pH values investigated. Marker formation during combined pressure-temperature (105 °C, 350 and 700 MPa) was influenced by both heat (which favored the marker formation) and pressure (which hindered marker formation). The net final concentration of the marker formed during PATP was higher than HPP, but lower than thermal treatments. This study suggests that 4-hydroxy, 5-methyl, 3(2H) furanone may not be a suitable marker for evaluating pressure–heat uniformity during PATP.