Ethyl-carbamate determination by gas chromatography–mass spectrometry at different stages of production of a traditional Brazilian spirit

Ethyl carbamate (EC), which is probably carcinogenic to humans, can be produced during the alcoholic fermentation of sugar-cane juice to give cachaça. The stages to produce cachaça are obtainment of sugar-cane juice, sugar-cane fermentation to wine, and obtainment of distilled fractions and residue. In order to investigate the presence of EC in the wine and in the fractions of the distillation process, as well as in the vinasse (the residue left after distillation), gas chromatography–mass spectrometry was employed. After the fermentation phase, the wine showed an average content of 122 mg L⁻¹ of EC. Average EC content in distilled fractions was 59.7 mg L⁻¹ for head, 52 μg·L⁻¹ for heart and 1.57 mg L⁻¹ for tail. EC content was 53.1 mg L⁻¹ for vinasse. The results showed that it is essential to separate the head and tail fractions to ensure cachaça quality, with respect to EC content.     

Determination of polycyclic aromatic hydrocarbons in cachaça by HPLC with fluorescence detection

Cachaça is a distilled alcoholic beverage obtained from sugar cane fermentation. In this study, 25 brands of cachaça commercially available in Brazil were analysed for the presence of 5 PAHs (benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene and dibenz[a,h]anthracene). The methodology involved liquid–liquid extraction with cyclohexane and dimethyilformamide–water (9:1, v/v), clean up on silica gel column and determination by high performance liquid chromatography using fluorescence detection. PAHs peak identity was confirmed by gas chromatography–mass spectrometry (GC–MS). Variable levels of summed PAHs were detected in the analysed samples, ranging from not detected to 1.94 μg/L. The results confirm the presence of PAHs in cachaça and suggest that contaminated sugar cane may be the source of PAHs in sugar cane spirits.     

可同化氮源磷酸氢二铵对发酵型猕猴桃酒品质的影响

为考察可同化氮源磷酸氢二铵（diammonium phosphate,DAP）对发酵型猕猴桃酒品质的影响,向猕猴桃汁中添加100～400 mg/L（氮质量计,下同）的DAP,并对发酵所得猕猴桃酒的基本理化指标、潜在有害醇类物质、营养组分和香气物质进行检测与分析。结果表明：添加100～400 mg/L的DAP均可以加快起酵后48 h内的乙醇发酵速度,并显著降低了猕猴桃酒中高级醇和甲醇等潜在有害醇类物质产量。特别是添加300 mg/L的DAP时,猕猴桃酒的总高级醇和甲醇产量分别下降了42.84%和20.1%;尽管添加DAP显著降低了酒中的氨基酸总量,但却对猕猴桃酒中的VC和总酚含量没有显著影响;添加DAP显著增加了猕猴桃酒的乙酸酯类和乙酯类香气物质含量,其中,添加300 mg/L的DAP可以显著增加乙酸乙酯、正己酸乙酯、正辛醛、庚醇和1-辛烯-3-醇等香气物质含量,从而使猕猴桃酒果香和花香较为浓郁。因此,添加适量的可同化氮源DAP可以提升发酵型猕猴桃酒的品质。     

Molecular mass distribution of dextran in Brazilian sugar and insoluble deposits of cachaça

The dextran molecular mass distribution profile in 77 sugar samples from Brazil and twelve insoluble deposits (alcoholic flocks) samples from sugared cachaças (Brazilian sugar cane spirit) is described in terms of number-average molecular mass M_n, weight-average molecular mass M_w, Z-average molecular mass M_z, and polydispersity. The analyses were performed by size-exclusion chromatography, using a refractive index detector. In most of the sugar samples, it was possible to identify two major groups of dextrans with M_w averages of 5 × 10⁶ and 5 × 10⁴ Da. Based on the evaluated parameters, the dextran distribution profile is about the same in samples analyzed over five seasons, and, therefore, it is likely that the Brazilian product pattern will not change very much over the years. In insoluble deposits from sugared cachaças, dextrans with M_w values in the order of the 10⁵ Da were the most frequent ones, being present in 58% of the samples.     

Probiotic beverage from cashew apple juice fermented with Lactobacillus casei

This study optimized the conditions of Lactobacillus casei NRRL B-442 cultivation in cashew apple juice, as well as, determined the proper inoculum amount and fermentation time. Moreover, it was investigated the survivability ability of L. casei in cashew apple juice during refrigerated storage (4 °C) for 42 days. The optimum conditions for probiotic cashew apple juice production were initial pH 6.4, fermentation temperature of 30 °C, inoculation level of 7.48 Log CFU/mL (L. casei) and 16 h of fermentation process. It was observed that the L. casei grew during the refrigerated storage. Viable cell counts were higher than 8.00 Log CFU/mL throughout the storage period (42 days). The values of lightness, yellowness and total color change increased and the values of redness reduced along the fermentation and refrigerated storage periods. The fermented juice with L. casei is a good and healthy alternative functional food containing probiotics. Cashew apple juice showed to be as efficient as dairy products for L. casei growth.     

Yeast species involved in artisanal cachaça fermentation in three stills with different technological levels in Pernambuco,Brazil

Both the taste and aroma of cachaça, an alcoholic beverage produced by yeast fermentation of sugar cane, are influenced by yeast metabolites and volatiles. The knowledge of yeast population dynamics during the fermentation process will help to establish the basis for quality control of alcoholic beverage. In the present work, the population dynamics of three fermentation processes, with differing levels of technological sophistication, were studied. Saccharomyces cerevisiae was found to be the dominant species, but Candida milleri (Candida humilis), Pichia caribbica, Pichia guilliermondii and Zygosaccharomyces fermentati (Lachancea fermentati) were also significantly involved. In addition, four new yeast species that are not represented in NCBI/EMBL nucleotide database were found. These yeasts were classified as Candida sp., Candida drosophilae-like, Candida ubatubensis-like and Zygosaccharomyces sp. In fermentation trails at laboratory scale, all species were found to contribute to the production of volatiles. Thus it is probable that product quality is strongly dependent on population dynamics during the fermentation process.     

Effect of fusel oil addition on volatile compounds in papaya wine fermented with Williopsis saturnus var. mrakii NCYC 2251

The impact of fusel oil addition on volatile compounds formation in papaya wine fermented with yeast Williopsis saturnus var. mrakii NCYC2251 was studied with a view to enhancing papaya wine aroma production. Time-course papaya juice fermentations were carried out using W. saturnus var. mrakii NCYC2251 with fusel oil added (0, 0.1 and 0.5% v/v). Fermentation characteristics in terms of yeast growth, Brix and pH changes were similar for all fermentations except for those added with 0.5% (v/v) fusel oil. The addition of 0.5% (v/v) fusel oil inhibited yeast growth. A wide range of volatile compounds were produced during fermentation including acids, alcohols, esters and aldehydes with esters being the most abundant volatile compounds produced. The addition of 0.1% (v/v) fusel oil reduced the production of undesirable volatiles such as ethyl acetate and acetic acid, while increasing the desirable volatiles production such as ethanol and acetate esters. This study suggests that papaya juice fermentation with W. saturnus var mrakii NCYC 2251 together with a low concentration of added fusel oil can be another means of modulating papaya wine aroma compound formation.     

Addition of amino acids to grape juice of the Merlot variety: Effect on amino acid uptake and aroma generation during alcoholic fermentation

The effects of adding selected amino acids (phenylalanine, alanine, aspartic acid and threonine) to grape juice on the generation of aroma compounds and on amino acid uptake were studied. The fermentation kinetics varied according to the quantities of amino acids added. The fermentations finished more quickly in supplemented juices and their alcoholic content was significantly higher than in the control (p < 0.05). Amino acids were consumed mainly in the first quarter of fermentation. Higher alcohol formation took place at the same time as ethanol formation: with more amino acids present in the medium, more phenyl ethanol (p = 0.01) and benzyl alcohol were formed while isoamyl alcohol production decreased. The contents of isoamyl and phenylethyl acetates, ethyl hexanoate and ethyl octanoate, as well as most fatty acids increased during the fermentation, reaching a maximum for 10% of ethanol; with higher alcoholic contents, their concentrations decreased.     

Effect of modifiers for As,Cu and Pb determinations in sugar-cane spirits by GF AAS

Palladium plus magnesium nitrates with and without Ir, Ru and W were evaluated for the simultaneous determination of As, Cu and Pb in cachaça by graphite furnace atomic absorption spectrometry. For 20 μL of sample, 5 μL Pd(NO₃)₂ and 3 μL Mg(NO₃)₂ dispensed together onto the Ir-coated platform of the THGA, analytical curves in the 0–30.0 μg L⁻¹ As, 0–1.50 mg L⁻¹ Cu and 0–60.0 μg L⁻¹ Pb were built up and typical linear correlation coefficients were always better than 0.999. The limit of detection was 1.30 μg L⁻¹ As, 140 μg L⁻¹ Cu and 0.90 μg L⁻¹ Pb. As, Cu and Pb contents in 10 cachaça samples agreed with those obtained by ICP-MS. Recoveries of spiked samples varied from 96% to 106% (As), 97% to 112% (Cu) and 92% to 108% (Pb). The relative standard deviation (n = 12) was typically 2.7%, 3.3% and 1.9%.     

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.)

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16°Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y_p/s), biomass (Y_x/s), glycerol (Y_g/s) and acetic acid (Y_ac/s), the volumetric productivity of ethanol (Q_p), the biomass productivity (P_x), and the fermentation efficiency (E_f) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E_f, Y_p/s, Y_g/s, and Y_x/s parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 μg/L), CAT-1 (80,317.01 μg/L), VR-1 (67,573.99 μg/L) and S. bayanus CBS 1505 (71,660.32 μg/L). The highest concentrations of ethyl esters were 454.33 μg/L, 440.33 μg/L and 438 μg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 μg/L) and higher alcohols (83,996.33 μg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Effects of initial ammonium ion concentration on pullulan production by Aureobasidium pullulans and its modeling

Pullulan fermentations by Aureobasidium pullulans with various initial ammonium ion concentrations were evaluated in a 2-L bioreactor. The results demonstrated that A. pullulans produced highest pullulan (23.1 g/L) when the initial ammonium sulfate was 7 at 5 g/L. The purity of produced pullulan was 94.6%. Seven gram per liter of ammonium sulfate produced more biomass due to the higher level of nitrogen source, but the pullulan-degrading enzyme activity was detected after the depletion of sucrose, which reduced pullulan concentration. From the results of fed-batch fermentation, addition of 10 g/L of sucrose suppressed A. pullulans from producing pullulan-degrading enzyme. Additionally, the modified-Gompertz equation demonstrated its generality to fit all pullulan production, biomass production, and sucrose consumption curves at three ammonium sulfate levels. After incorporating the degrading factor, a re-modified-Gompertz equation was obtained that can adequately describe the decrease of pullulan at the late fermentation stage.     

Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants

The effects of yeast supplementation on intake, production, and rumen fermentation characteristics have been widely studied, but results are inconsistent between different studies. A quantitative meta-analysis was applied to 110 papers, 157 experiments, and 376 treatments dealing with yeast supplementation in ruminants. The objective was first to highlight the major quantitative effects of live yeast supplementation on intake, rumen fermentation, and milk production, and second, to identify major differences in experimental conditions between studies that can affect the response to treatment. Some of these experimental conditions are referred to as interfering factors. Yeast supplementation increased rumen pH (+0.03 on average) and rumen volatile fatty acid concentration (+2.17 mM on average), tended to decrease rumen lactic acid concentration (−0.9 mM on average), and had no influence on acetate-to-propionate ratio. Total-tract organic matter digestibility was also increased by yeast supplementation (+0.8% on average). Yeast supplementation increased dry matter intake (DMI; +0.44 g/kg of body weight; BW), milk yield (+1.2 g/kg of BW), and tended to increase milk fat content (+0.05%), but had no influence on milk protein content. Dose effects of yeast supplementation, expressed as log₁₀ [1+(cfu per 100 kg of BW)], globally confirmed the qualitative effects observed in the first analysis. The positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the DMI level. It was negatively correlated with the level of dietary neutral detergent fiber (NDF). The positive effect of yeast supplementation on rumen volatile fatty acid concentration increased with DMI and crude protein levels. The positive effect of yeast supplementation on organic matter digestibility increased with the percentage of concentrate and NDF in the diet. The negative effect of yeast supplementation on lactic acid concentration tended to decrease when the DMI level and the percentage of concentrate in the diet increased. The effects of interfering factors were globally similar when either dose effect or qualitative effect of yeast was taken into account. Although rumen fermentation efficiency per se was not measured, these results suggest an improvement in rumen fermentation by yeast supplementation. This effect could, however, be modulated by several different factors such as DMI, percentage of concentrate or NDF in the diet, or species.     

高浓麦汁中添加红枣汁对酵母发酵性能及啤酒风味的影响

该实验研究了红枣汁不同添加量（10%、15%、20%、25%、30%、35%）对啤酒高浓酿造中酵母发酵性能及风味物质的影响。结果表明,红枣汁的添加能显著提高酵母的发酵性能,发酵过程中红枣汁添加组总CO2质量损失均高于对照组,发酵结束后乙醇产量显著增加（P＜0.05）,其中,30%红枣汁添加量促发酵效果最好,发酵度为88.18%,酒精度为11.30%vol,与对照组相比,分别提高了4.16%和4.63%。添加红枣汁可显著影响啤酒的色值：红枣汁添加比例越高,啤酒L*值越低,a*值与b*值越高,总色差（ΔE值）与对照组差异越大。与对照组相比,红枣汁的添加减少了高级醇的形成,显著降低了啤酒的醇酯比。     

Effect of dilution rate and nutrients addition on the fermentative capability and synthesis of aromatic compounds of two indigenous strains of Saccharomyces cerevisiae in continuous cultures fed with Agave tequilana juice

Knowledge of physiological behavior of indigenous tequila yeast used in fermentation process is still limited. Yeasts have significant impact on the productivity fermentation process as well as the sensorial characteristics of the alcoholic beverage. For these reasons a better knowledge of the physiological and metabolic features of these yeasts is required. The effects of dilution rate, nitrogen and phosphorus source addition and micro-aeration on growth, fermentation and synthesis of volatile compounds of two native Saccharomyces cerevisiae strains, cultured in continuous fed with Agave tequilana juice were studied. For S1 and S2 strains, maximal concentrations of biomass, ethanol, consumed sugars, alcohols and esters were obtained at 0.04 h⁻¹. Those concentrations quickly decreased as D increased. For S. cerevisiae S1 cultures (at D = 0.08 h⁻¹) supplemented with ammonium phosphate (AP) from 1 to 4 g/L, concentrations of residual sugars decreased from 29.42 to 17.60 g/L and ethanol increased from 29.63 to 40.08 g/L, respectively. The S1 culture supplemented with AP was then micro-aerated from 0 to 0.02 vvm, improving all the kinetics parameters: biomass, ethanol and glycerol concentrations increased from 5.66, 40.08 and 3.11 g/L to 8.04, 45.91 and 4.88 g/L; residual sugars decreased from 17.67 g/L to 4.48 g/L; and rates of productions of biomass and ethanol, and consumption of sugars increased from 0.45, 3.21 and 7.33 g/L·h to 0.64, 3.67 and 8.38 g/L·h, respectively. Concentrations of volatile compounds were also influenced by the micro-aeration rate. Ester and alcohol concentrations were higher, in none aerated and in aerated cultures respectively.     

Ethyl carbamate in cachaça (Brazilian sugarcane spirit): Extended survey confirms simple mitigation approaches in pot still distillation

In 2009, we reported an association between low levels of ethyl carbamate (EC) in pot still cachaças from Paraíba State, Brazil, and distillation in copper pot stills equipped with cooled columns. To strengthen these observations, we extended our study to Pernambuco State and assessed 13 pot still and 20 column still cachaça brands. An EC range from <40 to 532 μg/l was found; 18 brands exceeded the Brazilian limit (150 μg/l), 89% of which were column still types. Mean EC concentration of pot still cachaças was very low (64 μg/l), and was well below the Paraíba study (220 μg/l). An on-site investigation of pot still distilleries associated with <40 μg/l brands showed a connection to differences in the distillation apparatus. Maximising distillation reflux ratios in the ascending parts and minimising exposure to copper in the descending parts (through the use of stainless steel) can reduce EC, and also avoid copper contamination.     

Potato juice fermented with Lactobacillus casei as a probiotic functional beverage

This study was conducted to evaluate probiotic potato juice as a potential substrate for the production of Lactobacillus casei, and the change in the functionality of potato juice was monitored during fermentation. L. casei grew well in potato juice without nutrient supplementation, and lactic acid bacteria of fermented ‘Haryoung’ juice reached 1.7×10⁹ CFU/mL after a 48 h fermentation. DPPH radical scavenging activities of the potato juices decreased after a 72 h fermentation, but fermented colored potato juice still maintained >50% radical scavenging activity. The survival rate of L. casei fermented in’ Haryoung’ juice was 89.0% after exposure to an acidic condition, and L. casei in all fermented potato juice samples showed the ability (50–85%) to survive in the presence of bile. These results suggest that fermented potato juice might serve as a probiotic functional beverage for vegetarians or consumers who are allergic to dairy products.     

Influence of the distillation processes from Rio de Janeiro in the ethyl carbamate formation in Brazilian sugar cane spirits

Ethyl carbamate (EC) or urethane, a potentially carcinogenic substance found in significant amounts in distilled fermented beverages, has become the main technical obstacle to the exportation of Brazilian sugar cane spirits (cachaças). In this evaluation, the EC levels were measured in samples of recently distilled beverages and of finished products, collected at 28 production plants using different distillation systems, from various regions of the state of Rio de Janeiro. The results obtained through GC–MS, using selective monitoring of ions with m/z 62 and 74, showed that approximately 45% of the products and raw materials are above the maximum allowed value of 150 μg/l, with an EC average value of 160 μg/l. The lower values were those for samples from alembics using low temperature and high reflux rate distillation, which emphasized the role of these two variables on the EC levels.     

Juice matrix composition and ascorbic acid fortification effects on the phytochemical,antioxidant and pigment stability of açai (Euterpe oleracea Mart.)

The effects of juice matrix composition on the phytochemical stability of açai (Euterpe oleracea Mart.) were evaluated by contrasting natural clarified juice systems with isolated polyphenolic and anthocyanin fractions, in the presence or absence of ascorbic acid (500 mg/l) under accelerated storage conditions (37 °C). Polyphenolic (anthocyanin and non-anthocyanin polyphenolics) and anthocyanin fractions were isolated using C18 Sep-Pak columns and then re-dissolved in the original aqueous juice matrix (unbound fraction) or in a citric acid buffer (pH 3.5). The isolation of anthocyanins from the açai matrix improved their colour stability but a greater retention of non-anthocyanin polyphenolics and antioxidant properties was favoured by the initial juice composition. The presence of non-anthocyanin polyphenolics exerted a protective effect against ascorbic acid oxidation and enhanced polyphenolic and antioxidant stability in isolates fortified with ascorbic acid. However, all isolates obtained from açai experienced significant colour, polyphenolic, and antioxidant losses during storage and this indicated that optimization of early stages of industrial processing, storage and distribution are necessary to retain the functional properties of açai-containing products.     

Antimutagenic activity of Aspergillus awamori-fermented black soybean response to simulated digestive juice treatments and its antimutagenic mechanisms

Aspergillus awamori-fermented black soybeans, a potential healthy food ingredient, possess antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO); a direct mutagen and benzo[a]pyrene (B[a]P; an indirect mutagen). Results of this study revealed that antimutagenic, desmutagenic, and blocking effects all contributed to the antimutagenicity of the methanol of fermented black soybean. However, the desmutagenic effect of the fermented black soybean extract on B[a]P is not caused by the interaction of intact B[a]P or S9 mixture with the extract. Instead, it is due to the interaction of B[a]P metabolites with the antimutagenic factors in the extract. Exposure to simulated gastric juice (pH 1.2) for 30 min or intestinal juice for 2 h resulted in a reduced antimutagenicity of fermented black soybean extract. Nevertheless, the simulated gastric juice treated-fermented black soybean extracts and the simulated intestinal juice treated-fermented black soybean extracts still possessed a considerable antimutagenic effect against 4-NQO and B[a]P.