ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE AND AROMATIC RED RICE BRAN

Shochu distillery waste which had been exhausted by a vacuum-distillation procedure at low temperature (35–40°C) was used for secondary ethanol fermentation with the bran of aromatic red rice (Oryza sativa var. Indica, Tapol). The filtrate of the fermented mash made from kome shochu distillery waste and aromatic red rice bran had a characteristic wine-like red color, contained about 12% ethanol (v/v), and possessed a fine aroma that was fortified with the aromas of higher alcohols and volatile esters during secondary ethanol fermentation. A novel red alcoholic beverage was produced from the industrial by-product of shochu-making, kome shochu distillery waste and a by-product of Sekihan-cooking, aromatic red rice bran. The filtrate of the fermented mash had a characteristic absorbance at 530 nm. As the amounts of aromatic red rice bran in the mash were increased, the absorbance at 530 nm increased. Thus, a novel system utilizing shochu distillery waste, which is conventionally treated as wastewater, and aromatic red rice bran was established economically using a simple vacuum-distillation and secondary ethanol-fermentation procedure without the need for any complicated or expensive processes.     

PRODUCTION OF SHOCHU ON A COMMERCIAL SCALE FROM POST-DISTILLATION SLURRY BY A NEWLY DEVELOPED RECYCLING PROCESS

During commercial scale fermentation for the production of rice shochu using post-distillation slurry in place of the first-stage mash, it was difficult to stir the mash from the first to the third day because of the high viscosity of the mash. Shochu was therefore fermented with a two-step addition of cooked rice, adding rice on the first and third days, to reduce the viscosity of mash during the first few days. Even though the ethanol concentration of the mash after 9 d was 17.2 v/v% and was a little lower than that obtained in laboratory scale tests, the yield of pure ethanol was 473 ml kg^?1 of rice and this value was higher than the average value (450 ml kg^?1) when shochu is made in the standard manner on a commercial scale. The shochu produced on a commercial scale by the newly developed recycling process was very mild and smooth, and received high scores in sensory tests.     

Development of Efficient Shochu Production Technology with Long‐term Repetition of Sashimoto and Reuse of Stillage for Fermentation

In traditional shochu production with a long‐term repetition of sashimoto, the activity of yeast cells decreases, generally resulting in bacterial contamination problems. In this study, a pilot scale study was carried out to demonstrate a technique developed with a laboratory scale test, which improved the activity of the yeast in the first‐stage fermentation and the possibility of reuse of stillage for the fermentation to reduce the quantity of stillage from the distillation by 50%. The yeast cells were activated by aeration and stirring for several hours after sashimoto. The yeast cells maintained a high activity level during the entire test period. The ethanol concentration of the second‐stage fermented mash was improved and an ethanol concentration of more than 17.5% (v/v) was achieved when stillage was reused in place of water for the fermentation. Comparing the flavour compounds of shochu produced by the traditional process with the long‐term repetition of sashimoto, 0.2 ppm of furfural was detected in the shochu produced from the stillage reuse process. No other significant differences were found in the concentrations of the low‐, middle‐ and high‐boiling flavour compounds examined, nor in the concentrations of isoamyl acetate and β‐phenethyl acetate, which are desired flavour compounds in shochu.     

Behaviour of ethyl caproate during the production and distillation of ethyl caproate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐rich rice shochu by adding a cultured broth of a caproic acid‐producing bacterial (CAPB) consortium to the fermentation. When the CAPB consortium containing fermented mash was subjected to vacuum distillation, the distillation efficiency of ethyl caproate was up to ~300%, while the distillation efficiencies of the other flavour compounds and ethanol were <100%. The behaviour of ethyl caproate during the production and distillation of an ethyl caproate‐rich rice shochu was investigated and the results showed that ethyl caproate was synthesized by Saccharomyces cerevisiae during the shochu production process, and that some of the ethyl caproate synthesized was secreted into the medium. Ethyl caproate in the medium evaporated easily and was transferred to the distillate when distilled. The increase in ethyl caproate after distillation was mainly derived from the release of the intracellular ethyl caproate, with negligible input from the chemical esterification of caproic acid and ethanol. During vacuum distillation, although few yeast cells were disrupted, secretion of the intracellular ethyl caproate was more efficient owing to the increase in temperature, allowing the distillation efficiency of ethyl caproate to be >100%. Copyright © 2016 The Institute of Brewing & Distilling     

The Formation of β‐Damascenone in Sweet Potato Shochu

The formation of β‐damascenone during shochu manufacture was investigated by quantifying β‐damascenone at each stage of manufacturing. Steamed sweet potato has a low level of free β‐damascenone (0.02–0.1 μg/g). During fermentation, β‐damascenone was produced in small quantities that were degraded by yeast. Thus, the second mash accumulates little free β‐damascenone (approximately 17 μg/L). The concentration profile in the fractionated distillate showed that β‐damascenone was produced during heating. Most β‐damascenone in shochu was formed during distillation, not during steam heating and fermentation. It is suggested that the level of β‐damascenone in shochu could be increased by reducing the pH of the second mash and prolonging the distillation period. Sweet potato cultivars differed in total free and hydrolyzed β‐damascenone content and there was a strong association between each cultivar and its shochu β‐damascenone content. The selection of the sweet potato cultivar is important for determining the quantity of β‐damascenone in a shochu brew.     

Improving Bioethanol Yield: The Use of Solid‐State Fermentation Products Grown on DDGS

In a climate of uncertain prices for grain and fuel, conservation and efficiency are more important than ever. Many agro‐industrial residues are excellent substrates for solid‐state fermentation (SSF). Dried distillers grains with solubles (DDGS) are an important co‐product of the ethanol industry and have potential as a substrate for solid‐state fermentation. SSF products were produced using DDGS from a distillery and DDGS from a fuel ethanol plant as the fungal growth substrate. The spent grains were inoculated with GRAS (Generally Regarded As Safe) organisms Aspergillus orzyae and Rhizopus oligosporus. After growth, the resultant SSF product was dried and used as an enzyme complex supplement that was added to laboratory scale standard fuel ethanol corn mash fermentations. Improved ethanol yields were consistently observed. The use of solid‐state fermentation to produce unique enzyme complexes on DDGS offers a novel way to increase the value of the DDGS and to enhance bioethanol fermentation.     

Development of a process for producing ethyl caproate‐ and ethyl lactate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐ and ethyl lactate‐rich rice shochu. On a laboratory‐scale, there was a shochu production trial with Saccharomyces cerevisiae Y‐E. Caproic acid added in the second‐stage fermentation was esterified to ethyl caproate. Both ethyl caproate and ethyl lactate were produced by adding a caproic acid‐producing bacterial (CAPB) consortium and lactic acid bacterium (LAB) to the shochu production process. Yellow koji was more appropriate for producing a flavour‐rich shochu with the addition of a CAPB consortium and LAB than white koji. Optimal addition time for the CAPB consortium and LAB was on the first day of the second‐stage fermentation, judging from concentrations of ethyl caproate and ethyl lactate produced. Additional dosages of CAPB consortium and LAB positively affected the formation of ethyl caproate and ethyl lactate, respectively. Shochu production with the addition of 2% and 4% CAPB consortium led to ethyl caproate concentrations of 27.3 mg/L and 47.9 mg/L in genshu, respectively, and the shochu achieved the best sensory test score from the Japanese shochu brewery panellists. Copyright © 2015 The Institute of Brewing & Distilling     

Production of a high concentration of ethanol from potato tuber by high gravity fermentation

To produce a high concentration of ethanol from viscous potato tuber mash, potato tuber mash containing high contents of solids (28%) was prepared by grinding the potato tuber without the addition of water. The viscosity of the potato mash was reduced by using Viscozyme (0.1%) at 50°C for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90°C for 30 min and optimal conditions for the simultaneous saccharification and fermentation (SSF) of the potato mash for ethanol production were investigated using statistical methods. Using 2⁴ factorial design, saccharifying-enzyme and incubation temperature were found to be important factors. Using response surface methodology, the optimal saccharifying-enzyme dosage and incubation temperature were determined to be 1.45 AGU/g dry matter and 31.3°C, respectively. Under these optimal conditions for SSF, 14.92%(v/v) ethanol with 91.0% of theoretical yield was produced after 60 h, and all the starch was completely used up.     

酒精蒸馏废液全循环工艺研究

研究了酒精蒸馏废液全循环对玉米粉液化、糖化和发酵的影响,结果表明,当液化酶用量达到14U/g、糖化酶用量为120U/g(玉米粉)时,蒸馏废液对液化无影响,而对糖化有促进作用;蒸馏废液能一定程度地促进酵母的生长,提高酵母耗糖速率;蒸馏废液经过25次循环发酵,酒精平均浓度在15.07%,平均糖利用率91.7%。蒸馏废液全循环工艺,解决了发酵法酒精生产中的污染问题,实现了发酵法酒精清洁生产。     

EFFECTS OF PROTEOLYTIC ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The quality of rice wine made from uncooked, unpolished aromatic red rice grain was improved by use of a commercial preparation of acid protease from Aspergillus niger during ethanol fermentation. The fermentation rate of the mash which contained the acid protease was much higher than that of mash that did not contain the preparation of acid protease. The rice wine made from uncooked, polished aromatic red rice, which usually had a less acceptable aroma, was improved by use of the preparation of acid protease, and large amounts of isobutyl alcohol, n-propyl alcohol and ethyl acetate were detected in the resultant rice wine. By contrast, the quality of rice wine made from the bran fraction of aromatic red rice was not improved by the preparation of acid protease. The polished rice fraction of aromatic red rice was affected by the acid protease and the aromatic quality of the rice wine was improved. The aromatic characteristics of red rice wine made from cooked, unpolished aromatic red rice grains, which was rather inferior in terms of both aroma and color, were also improved by the addition of the preparation of acid protease during ethanol fermentation. Thus acid protease has beneficial effects on the production of aromatic red rice wine.     

Isolation and Characterization of Shochu Yeasts with Superior Brewing Ability from Shochu Mashes

Seven yeasts with superior ability in terms of alcohol fermentation and aromatic ingredient generation were isolated from 272 wild yeasts obtained from shochu mashes of shochu breweries. These seven yeasts were examined for their fermentation ability with rice and sweet potato using small scale of fermentation tests. Moreover, their thermotolerance was evaluated by growth tests and fermentation tests with barley koji. Among the isolated seven yeasts, the shochu yeast “MF062” was superior in the characteristics of fast fermentation, high alcohol production, aromatic ingredient generation and thermotolerance. It was named the “Heisei Miyazaki Yeast”. From the results of analyses of physiological and genetic characteristics of MF062, it was shown to be a Saccharomyces cerevisiae, but it showed different characteristics from the industrial yeasts used in shochu production. Using a sweet potato shochu brewing test, with 60 kg of raw materials and yeast MF062, it was confirmed that a high alcohol production yield and a high quality shochu could be obtained.     

Characteristics of aromatic compound production using new shochu yeast MF062 isolated from shochu mash

The effect of temperature on the production of aromatic compounds using Heisei Miyazaki yeast MF062 was compared between 10 industrial yeasts. All yeasts tested produced characteristic patterns of alcohols and esters in fermentation tests with rice‐koji at 20, 28 and 38°C. The concentration and composition in mature moromi with rice‐koji at 20, 28 and 38°C were almost the same as those with barley‐koji. Therefore, it was suggested that fermentation temperature is an important factor in the production of aromatic compounds. MF062 produced almost the same concentration of β‐phenethyl alcohol at both 38 and 28°C. The concentration was higher than that generated by the other 10 yeasts. MF062 produced higher concentrations of i‐butyl alcohol than the other yeasts at higher fermentation temperatures. Moreover, compared with the other yeasts, MF062 produced a lower concentration of acetate, which can give an off‐flavour in excess concentrations in shochu. The production of acetoin was divided into two groups – a high producing group and a low producing group – at all temperatures. MF062 belonged to the latter group and showed preferred characteristics in the production of shochu, resulting in a high concentration of preferred aromatic compounds and a low concentration of compounds that impart an off‐flavour. Copyright © 2013 The Institute of Brewing & Distilling     

Effect of various activators on the course of alcoholic fermentation

The aim of the paper was to study the effect of various activators, i.e. vitamins, mineral compounds as well as products of vegetable and microbiological origin, on the efficiency, dynamics and other biotechnological indices of rye mash fermentation and on the quality of the high wines obtained. Studies were carried out on the sweet rye mash (density 19.5 °Blg) brought from the agricultural distillery.It was found that the addition of calcium pentothenate to the rye mash resulted in the improvement of the high wines quality by limiting the amount of the formed aldehydes and higher alcohols and simultaneously, it had no effect on the fermentation process. Soybean flour turned out to be a good activator of fermentation and it improved the majority of biotechnological indices of the process. The only negative aspect while applying that activator was the formation of higher amounts of higher alcohols in high wines. A comparative analysis of all the variants allowed us to state that, although some activators limited the level of higher alcohols, they did not contribute to the reduction of the formed aldehydes and even caused that their amount permissible by Polish Standard was exceeded; it was observed when biotin, biotin with thiamine, magnesium sulphate and higher amounts of thiamine (above 0.1 g/dm³ of mash) were applied.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

SAKE BREWING FROM LIQUEFIED-RICE WITH IMMOBILISED FUNGAL MYCELIA AND IMMOBILISED YEAST CELLS

Sake brewing from liquefied-rice solution with immobilised fungal mycelia and immobilised yeast cells was investigated. Rice was liquefied by α-amylase in order to improve fluidity. Aspergillus oryzae was used for the production of saccharifying enzymes, and Saccharomyces cerevisiae for fermentation. The saccharifying enzyme productivity of immobilised fungal mycelia grown in highly aerobic conditions was much higher than that of fungal mycelia grown in liquid culture. Furthermore, saccharifying enzyme production was stimulated by the use of liquefied-rice treated with protease. The enzyme activity of immobilised mycelia was 4.5 times higher than that of rice-koji. The saccharifying enzyme was produced 10 times over a period of about 30 d at 41°C using protease treated liquefied-rice. Ethanol production was carried out with immobilised yeast using liquefied-rice containing saccharifying enzyme extracted from immobilised fungal mycelia. 19.0% (v/v) ethanol was obtained after incubation at 15°C for 5 d.     

木薯酒精蒸馏废液循环回用工艺的探讨

研究了木薯酒精发酵中,蒸馏废液回用比分别为30%(v/v),50%(v/v),70%(v/v)和90%(v/v)时对酒精发酵的影响,循环回用实验共进行11批。结果表明,循环回用比例为30%(v/v)时对酒精发酵略有影响;当循环回用比例≥50%(v/v)时,随着循环批次的增加,酒精产量下降。蒸馏废液中,酿酒酵母及污染杂菌产生的代谢副产物(如有机酸、甘油等)的累积是造成酒精产量下降的主要原因。而蒸馏废液中氨氮的累积造成了发酵液颜色的加深。     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

Effects of altering fermentation parameters on glycerol and bioethanol production from cane molasses

Current bioethanol production processes do not produce more than approximately 13 g l^?1 glycerol at a purity of 11% total solids in the fermented mash. Such quantities are not sufficiently high for economic exploitation unless very‐large‐capacity plants are involved. It was envisaged that the ratio of glycerol to bioethanol could be altered in favour of glycerol by adjusting such fermentation parameters as osmotic pressure (water activity), pH, temperature and yeast cell inoculum. At 39.9 ° brix, pH 8.7, 38 °C, 90.7 atm and 120 × 10⁶ yeast cells ml^?1, glycerol formation in fermented mash increased to 56 g l^?1 at a purity of 14% total solids. The potential for high glycerol production, hence higher revenue, has been demonstrated. Adoption of this technology could have a significant positive economic impact on existing or new biomass‐to‐ethanol plants. © 2002 Society of Chemical Industry     

Processing factors affecting the level of residual cyanohydrins in gari

Intake of cyanogens in gari, a food processed from cassava roots, is implicated in the causation of tropical ataxic neuropathy (TAN). This neurological syndrome is endemic in some communities in south‐western Nigeria. Studies have shown that methods of processing cassava roots determine the quantity of cyanogens in gari. This study was conducted to investigate the effects of the method of dewatering and the duration of fermentation on cyanogens in gari. Cassava roots (400 kg) were peeled, washed, grated and divided into 14 woven polyethylene sacks. The mash in seven of the sacks was dewatered continuously during fermentation, while the mash in the remaining seven sacks was fermented without dewatering, but dewatered at the end of fermentation. Cassava mash from each treatment was roasted into gari at 24 h intervals up to 168 h. Mean cyanohydrin content in gari roasted from cassava mash dewatered continuously during fermentation was 10.8 mg HCN eq kg^?1 dw (CI 9.7–11.9), while mean cyanohydrin content in gari roasted from cassava mash dewatered after fermentation was 6.3 mg HCN eq kg^?1 dw (CI 5.3–7.4). Mean linamarin content was 4.0 mg HCN eq kg^?1 dw (CI 3.1–4.9) and mean HCN content was 1.6 mg kg^?1 dw (CI 1.3–1.9) in gari roasted from cassava mash dewatered continuously, while mean linamarin content was 3.2 mg HCN eq kg^?1 dw (CI 2.3–4.0) and mean HCN content was 1.2 mg kg^?1 dw (CI 0.9–1.5) in gari roasted from cassava mash dewatered after fermentation. The method of dewatering cassava mash and the duration of fermentation were significantly associated with the level of cyanohydrin in gari (p < 0.001). This study shows that dewatering of cassava mash continuously during fermentation contributes to the dietary cyanide load in TAN‐affected communities. © 2002 Society of Chemical Industry