Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

Critical effect of oxygen on aroma compound production by Proteus vulgaris

Aroma compound production by Proteus vulgaris was studied in a liquid medium under two culture conditions, static or shaken. By using a dynamic headspace technique coupled with gas chromatography–mass spectrometry, 30 volatile components, aldehydes, alcohols, esters, ketones, sulphur compounds and other miscellaneous compounds were identified. P. vulgaris produced a variety of volatile compounds in different amounts, depending on its culture condition. Sensory analysis has shown that the aroma characteristics of each culture condition were clearly differently perceived. The volatile composition was in agreement with the aroma perception. The culture medium obtained without agitation was characterised by a sulphur/rotten egg note and by a high level of H₂S. The culture medium obtained with agitation was characterised by fruity/fresh and cheese notes and by a high level of alcohols and volatile sulphur compounds. Thus, P. vulgaris could be a useful microorganism to produce various and numerous natural aroma compounds, depending on the culture conditions during fermentation.     

Characterisation of bound volatile compounds of a low flavour kiwifruit species: Actinidia eriantha

Aroma compounds in fruit are known to occur in free and glycosidically bound forms. The bound volatile fraction of a low flavour kiwifruit species, Actinidia eriantha, was studied. The fruit have a bland and grassy flavour. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analysed by GC–MS. Alcohols, terpenoids and phenolics were the most numerously represented compound classes. Alcohols, benzenoids and phenolics showed the highest concentrations. Major compounds were 2-phenylethanol, furfuryl alcohol, (Z)-3-hexen-1-ol, coniferyl alcohol, isoamyl alcohol and linolenic acid. Several of the bound compounds found, including linoleic, linolenic and benzoic acids and coniferyl alcohol, are precursors of odorous volatiles. Many compounds detected as bound volatiles have not been previously reported as free volatiles in A. eriantha. The bound volatile composition of A. eriantha also showed differences with those of other kiwifruit species.     

Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.     

Screening of dairy yeast strains for probiotic applications

To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.     

First mass spectrometry metabolic fingerprinting of bacterial metabolism in a model cheese

Metabolic fingerprinting is an untargeted approach which has not yet been undertaken to investigate cheese. This study is a proof of concept, concerning the ability of mass spectrometry (MS) metabolic fingerprinting to investigate modifications induced by bacterial metabolism in cheese over time. An ultrafiltrated milk concentrate was used to manufacture model cheeses inoculated with Lactococcus lactis LD61. Metabolic fingerprints were acquired after 0, 8 and 48 h from two different fractions of the metabolome: the water-soluble fraction using liquid chromatography–high resolution-MS and a volatile fraction using gas chromatography–MS. Metabolic fingerprints differed significantly over time. Forty-five metabolites were identified, including well-known cheese metabolites, such as 12 amino acids and 25 volatile metabolites, and less studied ones, such as four vitamins, uric acid, creatine and l-carnitine. These results showed the relevance of cheese MS fingerprinting to generate new findings and to detect even slight differences between two conditions.     

Characterization of total capsaicinoids,colour and volatile compounds of Habanero chilli pepper (Capsicum chinense Jack.) cultivars grown in Yucatan

Total capsaicinoids, colour and volatile compounds of 10 Habanero chilli pepper (Capsicum chinense Jack.) cultivars grown in Yucatan, grouped by their colours: four red, five orange and one brown, were determined. The content of capsaicinoids, responsible for the pungency of chilli peppers, varied between 41.8 and 65.9 mg g⁻¹ dry fruit. Mean concentration of orange cultivars was 55.0 mg g⁻¹, while red cultivars had 45.0 mg g⁻¹ dry fruit, indicating that the first ones are more pungent. The composition of volatile compounds of the Habanero chilli peppers differs clearly for the different cultivars. Orange and brown cultivars have in general higher amounts of esters, with their fruity odour notes, than red cultivars. These differences are reflected in the amount of total volatiles, which is higher in orange and brown cultivars in comparison with red cultivars. From these results it can be concluded that orange and brown cultivars are better in terms of their flavour-relevant chemical composition than red cultivars.     

基于气相色谱-离子迁移法解析山桐子油挥发性风味物质成分及精炼过程中变化规律

目的 探究精炼过程中山桐子油挥发性风味物质成分的变化规律。方法 采用气相色谱-离子迁移法(gas chromatography-ion mobility spectrometry,GC-IMS)获取不同处理山桐子油样品挥发性化合物指纹图谱,利用多元统计法分析不同处理下山桐子油中的挥发性风味物质差异。结果 山桐子油中共检测出107种挥发性化合物,鉴定出95种,其中酸类物质6种、醇类物质20种、醛类物质30种,酯类物质10种,酮类物质16种、其他类物质13种。结论 精炼工艺可以有效去除山桐子毛油中刺激性异味,提高令人愉悦的风味物质含量,因此经过精炼后的山桐子油具有微甜的果香风味。但由于关键挥发性化合物种类较少、含量不高,精炼后山桐子油的风味整体偏淡。     

Cooperation between wild lactococcal strains for cheese aroma formation

Several wild lactococcal strains were tested for their ability to produce aroma compounds during growth in milk. Strains were incubated alone and in combination with Lactococcus lactis IFPL730, which is characterized by showing α-keto acid decarboxylase activity. Volatile compounds from incubated milks were analyzed by means of solid-phase microextraction (SPME) and evaluated by gas chromatography–mass spectrometry (GC–MS). Incubated milks were also sniffed for sensory analysis to describe aroma attributes. The combination of L. lactis IFPL326 that showed the highest branched chain aminotransferase activity with IFPL730 contributed to the highest formation of leucine-derived volatile compounds, such as 3-methylbutanal, 3-methyl-1-butanol and 2-hydroxy-4-methyl pentanoic acid methyl ester. In addition, the milk incubated with this combination of strains was awarded, by the test panellists, the highest scores for “ripened cheese” attribute and aroma intensity. The results indicate that combination of L. lactis strains harbouring complementary catabolic routes can contribute to improved cheese aroma formation, the combined cultures with L. lactis IFPL730 resulting in higher volatile compound formation than isolate strains.     

Volatile constituents of Cachucha peppers (Capsicum chinense Jacq.) grown in Cuba

The steam volatile components of three cultivars of Cachucha mature peppers (Capsicum chinense Jacq.) were isolated by steam-distillation-continuous-extraction and analysed using GC and GC-MS. The composition of volatile compounds of the peppers differs clearly for the different cultivars. The content of volatile compounds, responsible for the flavour of Cachucha peppers, varied between 110.71 and 302.53 mg kg⁻¹. One hundred and thirty-six compounds were identified, from which hexyl isopentanoate, hexyl pentanoate, hexyl 2-methylbutanoate, 3,3-dimethylcyclohexanol, γ-himachalene and germacrene D were the major ones.     

Sensory improvement of dry-fermented sausages by the addition of cell-free extracts from Debaryomyces hansenii and Lactobacillus sakei

The effects of the addition of a combined cell-free extract from Lactobacillus sakei and Debaryomyces hansenii (D+L) or just a D. hansenii cell-free extract (D) to the initial formulation of a dry-fermented sausage were evaluated. The differences found among batches in the main microbial populations, pH, moisture content and global proteolytic and lipolytic indexes (total free amino acids, non protein nitrogen, acidity and tiobarbituric acid index) were not significant. Only, the acidity value of batch D was significantly higher (p<0.05) than that of batch D+L. Thus, cell-free extract from D. hansenii accelerated the lipolysis. Moreover, there were some significant differences (p<0.05) in the amino acid profile and, especially, in the aroma profile. The combination D+L and D promoted the generation of volatile compounds derived from lipid oxidation and carbohydrate fermentation. In batch D, the production of volatile compounds derived from amino acid catabolism and microbial fermentation was also enhanced. The overall quality was improved by both treatments (D+L, D) and also the aroma by addition of the combination of extracts (D+L). It is concluded that the addition of cell-free extracts from D. hansenii and, particularly, D. hansenii plus L. sakei could be useful to improve the final quality of fermented sausages.     

SPME-GC-MS与SPME-GC-O协同鉴定菇娘果关键香气成分

运用顶空固相微萃取、气相色谱-质谱联用、气相色谱-嗅闻（gas chromatography-olfactometry,GC-O）技术,结合气味活性值（odor active value,OAV）对菇娘果挥发性成分和香气活性成分进行鉴定,综合评价单个挥发性组分对整体香气贡献程度,确定关键香气成分。结果表明,菇娘果中有43 种挥发性组分,其中有10 种主要香气活性物质（OAV＞1）,主要包括4 种醛酮类化合物（正己醛、甲硫基丙醛、反-2-顺-6-壬二烯醛、1-辛烯-3-酮）,其香气分别描述为青草味、薯片味、黄瓜味、蘑菇味;5 种酯类化合物（甲基-2-甲基丙酸酯、丁酸甲酯、2-甲基丁酸甲酯、丁酸甲基乙基酯、癸酸甲酯）,其香气分别描述为花香、果香、苹果香、刺激果香、葡萄酒香;1 种呋喃类化合物（2-戊基呋喃）,其香气描述为焦糖味。GC-O法中,4 名嗅闻人员主要鉴定出“苹果味”2-甲基丁酸甲酯、“薯片味”甲硫基丙醛和“青草味”己醛。通过香气定性和感官定量描述性分析发现,2-甲基丁酸甲酯、甲硫基丙醛和己醛是菇娘果的主要香气成分。     

Changes in volatile compounds of Habanero chile pepper (Capsicum chinense Jack. cv. Habanero) at two ripening stages

The steam volatile components of Yucatan Habanero chile pepper (Capsicum chinense Jack. cv. Habanero) at two ripening stages (green and orange) were analyzed using GC and GC/MS. Both samples had several compounds in common. One hundred and two compounds were identified, from which (E)-2-hexenal, hexyl 3-methylbutanoate, (Z)-3-hexenyl 3-methylbutanoate, hexyl pentanoate, 3,3-dimethylcyclohexanol, and hexadecanoic acid were found to be the major constituents. During Habanero chile pepper maturation, the majority of volatile compounds decreased or even disappeared, some of them with green odour notes while esters, which have fruity odour notes, increased at the same time.     

Yeasts from Water Buffalo Mozzarella, a traditional cheese of the Mediterranean area

Countries of the Mediterranean area are characterized by production of artisanal cheeses, obtained from goat, sheep, cow and buffalo raw milk. The numbers and species of yeasts in the different cheeses are variable, but some species are more frequently detected than others. Kluyveromyces marxianus, K. lactis with their anamorph, Candida kefir, Debaryomyces hansenii and C. famata, C. colliculosa and C. catenulata are dominant species in several cheeses. However, Saccharomyces cerevisiae is often detected in pasta filata cheeses, such as Water Buffalo Mozzarella (WBM) or Cacio Cavallo Podolico. Recently, a comprehensive study of yeasts isolated from Mozzarella cheese produced in Basilicata (Southern Italy) has been carried out. The study has focused on lactose and/or galactose fermenting species (Kluyveromyces and Saccharomyces) to evaluate their role on the functional and sensory properties of the product. End products in milk were evaluated and the biodiversity in terms of production of sulphur dioxide, higher alcohols, ethyl acetate, and acetaldehyde was studied. In particular, S. cerevisiae strains from Water Buffalo Mozzarella cheese, compared to strains isolated from different habitats, such as wine, exhibited considerable difference in the production of some volatile compounds. The diversity observed could be related to the particular microhabitat of S. cerevisiae occurring in whey cheese of water buffalo milk.     

Olfactometric characterization of aroma active compounds in fermented fish paste in comparison with fish sauce,fermented soy paste and sauce products

Aroma active compounds in commercial fermented fish meat paste product (fish miso), fermented soy paste (soy miso), fish sauce and soy sauces were characterized by using a dynamic headspace method for volatile isolation and GC olfactometry for odor perception. A total of 123 volatile compounds consisting mainly of aldehydes, alcohols, esters, ketones, furans, sulfur and nitrogen-containing compounds, aromatics and acids were consistently identified. A major 16 odor-active compounds were distinguished to contribute as key aroma compounds for the miso and sauce products. Olfactometric and sensory findings clearly differentiated miso products with caramelic, fruity aroma notes, whereas fish sauce products were characterized by ammoniacal, fishy, nutty and cheesy odor note. Soy sauce products, however, were dominated by nutty and cheese aroma. Use of koji for fish miso production was found effective to enhance sweet aroma to the product with a reduction of nutty, meaty and rancid nuance. Principal component analysis employed for statistical interpretations clearly elucidated the relationship among different types of fermented products.     

基于快速成熟模型的藏灵菇发酵切达干酪挥发性风味物质分析

通过建立快速成熟干酪模型,采用固相微萃取法提取传统藏灵菇发酵的切达干酪模型与商品发酵剂制作的切达干酪模型中挥发性成分,并结合气相色谱-质谱联用技术和气相色谱-嗅闻技术对萃取成分进行鉴定,结果表明醇类和酯类是藏灵菇发酵切达干酪成熟过程中的主要风味物质。藏灵菇发酵切达干酪模型中风味物质的种类和含量都明显高于商业发酵剂制作的切达干酪模型,其中酯类物质的变化最为显著。感官评价和风味分析结果表明,藏灵菇发酵切达干酪模型中酯类和醇类物质种类和含量更为丰富,风味更强,水果香味更浓郁,还具有酒香味。