Volatile compound generation in dry fermented sausages by the surface inoculation of selected mould species

The effect of the inoculation of dry fermented sausage surface with an atoxigenic, proteolytic and lipolytic strain of Mucor racemosus, Penicillium aurantiogriseum and Penicillium camemberti on the volatile composition was studied. The analysis of the headspace volatile compounds using gas chromatography/mass spectrometry enabled the identification of 55 volatiles. The study showed that every mould species produced a different volatile profile which was also different from that of the control sausages. Compounds derived from amino acid catabolism, i.e. branched aldehydes and the corresponding alcohols, were produced in higher amounts in sausages inoculated with Penicillium spp. On the other hand, volatiles coming from the microbial esterification were related to sausages inoculated with M. racemosus. The development of the fungal mycelia on the sausage surface protected lipids from oxidation, thus giving rise to fewer lipid oxidation products in the inoculated sausages.     

食盐添加量对哈尔滨风干肠脂质和蛋白氧化及挥发性化合物形成的影响

研究不同食盐添加量(2.5%、2.0%、1.5%、1.0%)对哈尔滨风干肠在发酵过程中脂质及蛋白氧化的影响。结果表明,降低食盐添加量可显著降低风干肠过氧化物值以及肌原纤维蛋白和肌浆蛋白的羰基含量和表面疏水性,增加总巯基含量(P<0.05),说明NaCl对风干肠的脂质和蛋白氧化有促进作用。此外,挥发性化合物分析结果表明,各组风干肠中挥发性化合物的含量随着发酵时间的延长逐渐增加,且食盐添加量对多数化合物的生成具有显著影响(P<0.05)。随着食盐添加量的降低,醛类物质总体水平下降,酮类、酸类、酯类和醇类物质的总体水平上升。与对照组(2.5%)相比,添加2.0%食盐的样品减少了因脂质和蛋白氧化生成的醛、醇、酸类等物质,增加了微生物代谢产生的酮、醇、酸、酯类等物质,改善了风干肠的整体风味。     

The contribution of Penicillium aurantiogriseum to the volatile composition and sensory quality of dry fermented sausages

An atoxygenic, proteolytic and lipolytic strain of Penicillium aurantiogriseum was tested for its ability to accelerate the production of volatile compounds and to improve the sensory properties of dry fermented sausages. The following batches of sausages were prepared: control; superficially inoculated with a spore suspension; added with an intracellular cell free extract; and superficially inoculated and added with the intracellular cell free extract. Higher levels of lipid oxidation products were found in the aroma extracts of sausages without a mould cover. In contrast, branched aldehydes and alcohols presented higher concentrations in superficially inoculated and extract added sausages, while esters only showed higher concentration in the first ones. The sensory analysis showed that sausages prepared with both treatments received the highest scores in all the properties evaluated, which demonstrated both the potential of this mould as producer of volatile compounds and the effectiveness of combining both treatments.     

Contribution of a selected fungal population to proteolysis on dry-cured ham

The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids. Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds. Several months of ripening time are required to allow the particular flavour to develop. The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages. The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham. For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures. Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams. Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing. However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams. In addition, the concentration for some of the more polar free amino acids (i.e. Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles. These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham.     

功能性发酵剂对发酵香肠氧化稳定性及挥发性风味物质的影响

以功能性发酵剂植物乳杆菌（Lactobacillus plantarum CD101）和模仿葡萄球菌（Staphylococcus simulans NJ201）为混合发酵剂制作发酵香肠,同时以商业发酵剂和自然发酵为对照。结合过氧化值（peroxide value,POV）、硫代巴比妥酸反应物（thiobarbituric reactive substances,TBARS）值、羰基值、巯基值和挥发性风味物质等探究功能性发酵剂对发酵香肠脂肪和蛋白质氧化以及风味的影响。结果表明：在香肠加工和贮藏过程中,随着时间的延长,不同处理组香肠的POV、TBARS值、羰基值总体呈现上升趋势,巯基值呈现下降趋势。接种功能性发酵剂制作发酵香肠在一定程度上能够抑制产品的脂肪氧化和蛋白氧化,显著提升挥发性风味物质种类（P＜0.05）,其抑制脂肪氧化的能力显著优于商业发酵剂（P＜0.05）,抑制蛋白氧化的能力与商业发酵剂效果相当。     

脂肪添加量对发酵香肠脂质和蛋白质氧化及挥发性化合物形成的影响

通过研究不同脂肪添加量（5%、10%、15%和20%）的香肠在发酵过程中脂质和蛋白质的氧化水平,并对发酵前后的挥发性化合物进行分析,最后对香肠进行感官评价。结果表明,随着脂肪添加量的增加,硫代巴比妥酸值显著增加,肌原纤维蛋白的羰基含量增加,总巯基含量降低（P＜0.05）,说明增加脂肪添加量会促进发酵香肠中脂肪和蛋白质的氧化;挥发性化合物分析可知,脂肪添加量较高的发酵香肠,因脂质自动氧化产生的醛、酮、酸、醇、酯和烷烃类化合物含量也相应增加（P＜0.05）;感官评价结果表明,脂肪添加量为15%的发酵香肠,其整体可接受性最佳（P＜0.05）。     

Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages

Debaryomyces hansenii strains, M4 and P2, isolated from natural fermented sausages were inoculated in slow fermented sausages to study their effect on processing parameters, microbial population, volatile compound and sensory characteristics. The inoculation of D. hansenii strains, M4 and P2, did not affect the ripening process as no differences in pH and Aw were detected. The dominance of the inoculated yeast strains along the process was followed by RAPDs of M13 minisatellite. The inoculated yeasts, P2 and M4, were recovered at the end of the ripening process although P2 appeared in higher counts than M4. The sausages inoculated with P2 resulted in a decrease in lipid oxidation values (TBARS) and a reduction of lipid-oxidation derived aldehydes in addition to a highest acid compound abundance. M4 inoculated sausages resulted in highest sulphur containing compound abundance. However, no differences in consumer acceptance were detected. Moreover, both yeast strains were responsible for the generation of ethyl methyl-branched ester compounds in the dry-cured sausages.     

The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P < 0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24 × 10⁶ area units, respectively), alcohols for D. hansenii (363.77 × 10⁶ area units) and esters for C. zeylanoides (248.33 × 10⁶ area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P < 0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42 × 10⁶ area units) in comparison with non-inoculated batch (105.99 × 10⁶ area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.     

Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages

An atoxigenic strain of Penicillium camemberti was superficially inoculated on fermented sausages in an attempt to improve their sensory properties. The growth of this mould on the surface of the sausages resulted in an intense proteolysis and lipolysis, which caused an increase in the concentration of free amino acids, free fatty acids (FFA) and volatile compounds. Many of these were derived from amino acid catabolism and were responsible for the "ripened flavour", i.e. branched aldehydes and the corresponding alcohols, acids and esters. The development of the fungal mycelia on the surface of the sausages also protected lipids from oxidation, resulting in both lower 2-thiobarbituric acid (TBARS) values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols. The sensory analysis of superficially inoculated sausages showed clear improvements in odour and flavour and, as a consequence, in the overall quality of the sausages. Therefore, this strain is proposed as a potential starter culture for dry fermented sausage production.     

The influence of nitrite and nitrate on microbial, chemical and sensory parameters of slow dry fermented sausage

Nitrate and/or nitrite are used in the manufacture of dry-fermented sausages. However, research has mainly been focusing on nitrite and its effect on flavour development whereas little attention has been paid to nitrate. The aim of the present work was to study the effect of nitrate and nitrite as curing salts on the quality of a slow fermentation process. Two different batches containing nitrate or nitrite were manufactured. Microbial and chemical parameters were monitored during ripening and after vacuum packed storage, as well as their fatty acid composition and their profile of volatile compounds. The oxidation, measured as TBARS (Thiobarbituric acid reactive substances), was greater in the samples with added nitrite than in the samples with added nitrate. FFA (free fatty acids) release was higher in the samples containing nitrite throughout the process. Volatile compounds arising from amino acid degradation and carbohydrate fermentation were generated at higher levels in the samples with added nitrate, probably due to the higher population of microorganisms in these samples and the effect of nitrate on their metabolism.     

模仿葡萄球菌接种对发酵香肠品质及氧化稳定性的影响

以模仿葡萄球菌(SStaphylococcus simulans)NJ201接种发酵香肠为实验组,自然发酵香肠为对照组,测定香肠的pH值、色差、质构、过氧化值(peroxide value,POV)、硫代巴比妥酸反应物(thiobarbituric reactive substances,TBARS)值、羰基含量、总巯...     

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.     

Effect of selected mould strains on the sensory properties of dry fermented sausages

The effect of one Mucor and two Penicillium strains (Penicillium-3 and Penicillium-6) on the development of the sensory properties of dry fermented sausages has been studied. These strains were previously isolated from Spanish fermented sausages and selected for their proteolytic and lipolytic activity. Several experimental batches were prepared: one of them was non-inoculated and considered as control batch, three were inoculated with the selected strains, and the last one was inoculated with a commercial starter culture of P. nalgiovense. The pleasantness of the sensory attributes of the different batches were determined using an unstructured line scale of 10 cm. A triangular test was also carried out. Apart from their external appearance, batches inoculated with Mucor and Penicillium-3 had the highest scores for all the sensory attributes studied. The batch inoculated with P. nalgiovense had the lowest scores and similar to the control batch but its external appearance was considered to be the most pleasant. The batch inoculated with Penicillium-6 presented intermediate characteristics. A relation between these results and the enzymic activity of the moulds was established. The three strains selected for this study are proposed to be incorporated in a commercial starter culture because they improve the sensory properties of these meat products.     

亚硝酸钠对自然发酵哈尔滨风干肠微生物生长、脂肪氧化及挥发性化合物的影响

研究不同添加水平的亚硝酸钠（NaNO2）对自然发酵哈尔滨风干肠微生物生长、脂肪氧化和挥发性化合物的影响。结果表明,添加NaNO2对风干肠发酵过程中的pH值、水分活度、总菌落数和乳酸菌菌落数无显著影响（P＞0.05）,但添加0.10?g/kg以上的NaNO2可以显著降低风干肠中大肠杆菌菌落数（P＜0.05）。色差分析表明当NaNO2添加量达到0.10?g/kg时可以起到良好的发色作用,过氧化物值和硫代巴比妥酸反应产物显示NaNO2具有良好的抗氧化作用,NaNO2残留量随发酵时间的延长而逐渐降低,发酵结束后,各实验组间差异不显著（P＞0.05）。与此同时,挥发性化合物的分析结果显示,NaNO2添加量不影响风干肠中由碳水化合物发酵和氨基酸降解所产生的挥发性化合物的相对含量（P＞0.05）,但显著影响由脂肪氧化所产生的醛类和酮类化合物的相对含量（P＜0.05）,NaNO2添加量越大,对脂肪氧化产物的抑制作用越显著。     

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.     

Effect of fat content on aroma generation during processing of dry fermented sausages

Dry fermented sausages with different fat contents were produced (10%, 20% and 30%). The effect of fat content and ripening time on sensory characteristics, lipolysis, lipid oxidation and volatile compounds generation was studied. Also, the key aroma components were identified using gas chromatography (GC) and olfactometry. High fat sausages showed the highest lipolysis and lipid oxidation, determined by free fatty acid content and thiobarbituric acid reactive substances (TBARS), respectively. A total of 95 volatile compounds were identified using SPME, GC and mass spectrometry (MS). Fat reduction decreased the generation of lipid derived volatile compounds during processing while those generated from bacterial metabolism increased, although only at the first stages of processing. The consumers preference in aroma and overall quality of high and medium fat sausages was related to the aroma compounds hexanal, 2-nonenal, 2,4-nonadienal, ethyl butanoate and 1-octen-3-ol which contributed green, medicinal, tallowy, fruity and mushroom notes.     

Dominance and influence of selected Saccharomyces cerevisiae strains on the analytical profile of craft beer refermentation

This study investigated the influence of Saccharomyces cerevisiae strains inoculated at the refermentation phase on the analytical profile of craft beer. After preliminary screening of 33 S. cerevisiae strains, four were selected and used in bottle refermentation trials. To attribute the flavour profile of the refermented beers to the inoculated strains, molecular characterization was carried out using a mini‐satellite protocol of amplification with inter‐δ primers. Fingerprinting analysis of 500 isolates showed that all of the inoculated strains used in the bottle refermentation dominated the process. The main analytical characters across the bottle refermentation trials were similar, while there were distinctive and significant variations in the volatile compounds. In particular, when compared with the starter strain used as control, the bottle refermentation trials using the DBVPG 2170, DBVPG 2187 and L951 S. cerevisiae strains showed significantly higher levels of the volatile compounds that are responsible for fruity and flowery aromas (i.e. isoamyl acetate, ethyl octanoate, ethyl dodecanoate phenyl ethyl acetate, β‐phenyl ethanol). This study indicates that the proper refermentation process results in the dominance of the selected starter strain, which produces fermentation compounds that show a specific analytical profile and give the craft beer its distinctive bioflavour. Copyright © 2014 The Institute of Brewing & Distilling     

不同工艺条件下模仿葡萄球菌YD25对发酵香肠挥发性化合物的影响

以从如皋火腿中分离得到的一株模仿葡萄球菌(Staphylococcus simulans)YD25为研究对象,采用气质联用法研究不同肥瘦比、糖添加量及后熟时间条件下的发酵香肠中挥发性化合物种类及含量的变化情况,分析模仿葡萄球菌YD25对发酵香肠中挥发性化合物的作用效果。结果表明,在肥瘦比1:9(m/m)、糖添加量1%、后熟时间14d时,发酵香肠中检测到的挥发性化合物种类较多,分别为61、69、74种,而挥发性化合物的含量比例因不同工艺条件而发生相应的变化。     

基于电子鼻的红肠风味评价

为应用电子鼻快速、客观地评价红肠风味,使用电子鼻以及顶空固相微萃取和气相色谱-质谱联用对3 个不同生产批次的各批次3 种不同红肠中挥发性物质进行检测分析,并对其进行感官指标评定。采用支持向量机方法对不同红肠样品的电子鼻数据进行识别分类;利用相对气味活度值确定不同红肠中的关键风味物质;用主成分分析方法对红肠挥发性物质整体分析;利用正交偏最小二乘判别分析方法对电子鼻传感器和红肠关键风味的相关性进行分析,并使用逐步回归建立电子鼻与关键风味物质和感官评价指标数据的回归模型。通过支持向量机分析结果可知,电子鼻对不同种类红肠以及不同生产批次同种类红肠均具有良好的区别能力;气相色谱-质谱联用检测出不同种类化合物共117 种;通过相对气味活度值可知不同红肠中的关键风味物质种类差异较大,仅烯丙硫醇是各红肠的共有关键风味物质;主成分分析表明不同种类红肠在总体风味成分上明显不同;感官分析表明不同红肠在不同口感风味评价上存在差异,烟熏味和咸味为红肠最主要的风味;正交偏最小二乘判别分析显示电子鼻传感器数据与关键风味物质具有良好的相关性;建立电子鼻与关键风味物质和感官评价指标回归模型（R2＞0.8,P＜0.01）表明应用电子鼻可以对红肠风味进行评价预测分析。     

Aminogenesis control in fermented sausages manufactured with pressurized meat batter and starter culture

The application of high hydrostatic pressure (200MPa) to meat batter just before sausage fermentation and the inoculation of starter culture were studied to improve the safety and quality of traditional Spanish fermented sausages (fuet and chorizo). Higher amounts of biogenic amines were formed in chorizo than in fuet. Without interfering with the ripening performance in terms of acidification, drying and proteolysis, hydrostatic pressure prevented enterobacteria growth but did not affect Gram-positive bacteria significantly. Subsequently, a strong inhibition of diamine (putrescine and cadaverine) accumulation was observed, but that of tyramine was not affected. The inoculated decarboxylase-negative strains, selected from indigenous bacteria of traditional sausages, were resistant to the HHP treatment, being able to lead the fermentation process, prevent enterococci development and significantly reduce enterobacteria counts. In sausages manufactured with either non-pressurized or pressurized meat batter, starter culture was the most protective measure against the accumulation of tyramine and both diamines.