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.     

The Measurement of Structural Characteristics of Barley for Shochu Using Single‐Kernel Characterization System 4100 Crush‐Response Profiles

The selection of grain that is optimum for barley Shochu is not straightforward. Usually malting barley is used, but not all malting barley samples perform well in Shochu production. In addition there is a genotype × environment effect, so that a given variety may perform satisfactorily in one environment but not another. Therefore a method for testing samples for Shochu is required. This group has previously reported on the evaluation of barley for Shochu production using the Single Kernel Characterization System (SKCS) and demonstrated a significant correlation between the SKCS Hardness Index and pearling performance. The Hardness Index values are multivariate statistical predictions derived from an incremental change analysis of the crush‐response profiles. In the present study, a detailed analysis was performed of the averaged SKCS Crush‐Response Profiles of barley, obtained when measuring the SKCS Hardness Index. It was concluded that the morphological differences between averaged barley SKCS Crush‐Response Profiles appear to allow barley varieties to be classified into four distinct classes. In addition, the elastic response to the crushing on the SKCS device of the aleurone layers of barley kernels yields a useful predictor of pearling quality/performance.     

SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

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.     

Relationship Between Quality Parameters and SKCS Hardness Index in Malting Barley

To make an efficient and laborsaving quality selection in a malting barley breeding program, the relationship between malting quality parameters and the hardness index (HI) measured by Single Kernel Characterization System (SKCS) 4100 was studied with barley genetic resources and a group of descendant lines from “Yon Rkei 1363” that possess the high lysine allele lys1 and a very high diastatic power (DP). There was a highly significant correlation between the grain HI and the malt extract both in the genetic resources (r = ?0.48^**) and in the lys1 parent‐derived lines (r = ?0.50^**). Malt HI showed a highly significant correlation with malt extract (r = ?0.70^** in genetic resources, r = ?0.57^** in the lys1 parent‐derived lines) and malt total protein (r = 0.73^**, 0.84^**, respectively). For the selection of very high DP lines from the lys1 parent‐derived group, high grain HI selection was effective as all the very high DP lines had much harder grain characteristics. Therefore SKCS was regarded as a useful tool for quality selection in malting barley breeding.     

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     

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     

Classification of Barley Shochu Samples Produced Using Submerged Culture and Solid‐state Culture of Koji Mold by Solid‐phase Microextraction and Gas Chromatography‐Mass Spectrometry

An easy and fast method of analyzing volatile components contained in shochu, by headspace solid‐phase microextraction and gas chromatography‐mass spectrometry, was established to determine the difference in the content of the various components of barley shochu produced using a submerged culture of koji mold (submerged‐culture shochu) and a solid‐state culture of koji mold (solid‐culture shochu). The contents of the volatile components between the two types of shochu were compared. The results of the comparison revealed that the content of ethyl lactate, ethyl benzoate, 3‐methyl‐1‐pentanol, diethyl succinate, citronellol, and 2‐phenethyl acetate differed between the submerged‐culture and the solid‐culture shochu samples. In addition, the classification of barley shochu samples, including those on the market, into submerged‐culture shochu and solid‐culture shochu, was carried out by multivariate analysis using the quantitative values of the above‐mentioned six compounds, and distinct discrimination was found possible.     

Characteristic odour compounds in shochu derived from rice koji

Shochu, a traditional Japanese distilled liquor, makes use of rice koji, which is koji mould grown on rice grain. Rice koji is an essential ingredient of Japanese liquors such as shochu, and it plays a role as a source of the enzyme to degrade starch. However, there has been no research on the effect of rice koji on the flavour of shochu. Therefore, in this study, the volatile compounds in shochu derived from rice koji were investigated. Two shochu samples were prepared to assess the contribution of rice koji to the flavour. One shochu sample was prepared from rice koji, yeast and water (rice koji‐shochu). The other shochu sample was obtained from steamed rice and various enzymes instead of rice koji (enzyme‐shochu), along with yeast and water. The volatile compounds were analysed by gas chromatography–mass spectrometry (GC‐MS) and GC‐MS/olfactometry with aroma extract dilution analysis. The results showed that enzyme‐shochu had a higher flavour dilution value of dimethyl trisulphide and hexanal, whereas rice koji‐shochu had a higher flavour dilution value of some ester compounds that imparted aromatic odours such as fruity. Some unknown peaks representing compounds that impart characteristic odours such as soda, potato, lavender, and tea‐like were specifically detected in rice koji‐shochu. The concentrations and calculated odour active values of 14 compounds were measured. These results showed that isovaleraldehyde, ethyl caprylate, ethyl caproate and ethyl 2‐methylbutyrate played an important role in imparting the specific odour of rice koji‐shochu. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

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.     

Measurement of Barley Grain Rheology for the Quality Selection of Breeding Material

The Single‐Kernel Characterization System (SKCS) 4100 was used to test whole grain barley from a replicated breeding trial across two environments and two seasons. Hardness was calculated from a crush response profile of the resistance to shell and endosperm crushing as a function of time. This rheological data recovered was averaged over 300 grains for each sample then partitioned into genetic, environmental and error components in order to calculate the genetic components and heritability. The results demonstrated a high level of genetic variance for all rheological parameters at all sites and years. In addition, the heritability was also moderate to high (68% to 90%) for the SKCS hardness and the rheological parameters that had been previously shown to provide information about endosperm modification during malting. Since the rheological information is recovered for individual grains within a sample, the SKCS 4100 may provide a new tool for screening breeding material for uniformity of quality.     

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.     

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     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.     

PREDICTION OF FLOUR EXTRACTION RATE IN HARD RED WINTER WHEAT USING THE SINGLE KERNEL CHARACTERIZATION†

Flour extraction rate is a key determinant in milling efficiency and profitability and can be useful in projecting flour output from a mill. Wheat is generally purchased based upon a small group of traditionally measured physical characteristics. This article explores the use of the Single Kernel Characterization System (SKCS) to predict flour extraction rate. Regression analysis was performed using the SKCS parameters and test weight against flour extraction rate for over 600 observations from multiple years from the hard red winter wheat production areas of the U.S. The regression equation had an R²of 0.81. The data suggest that the SKCS 4100 and test weight can be used to predict flour extraction rate in hard red winter wheat. Using the regression equation as a tool, mill buyers may be able to make better decisions regarding their wheat purchases and prediction of flour output.     

Part III: the influence of serial repitching of Saccharomyces pastorianus on the production dynamics of some important aroma compounds during the fermentation of barley and gluten‐free buckwheat and quinoa wort

The present paper is the last report of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat or quinoa fermentation medium. In particular, it focuses on the production dynamics of important volatile compounds typically associated with the aroma of beer. Samples were taken every 24 h after 11 serial repitchings of a single starter culture, analysed for the particular aroma compound content by distillation followed by gas chromatography with flame ionization detection. The term ‘serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Results showed that the levels of methanol in the quinoa wort fermentation were only slightly higher than in barley and in practical terms independent of successive fermentation. The behaviour of acetaldehyde in quinoa was similar to that in barley. However, there was a final 2‐fold lower production of some important aroma compounds compared with barley and buckwheat and for this reason quinoa cannot be recommended as a gluten‐free substitute to produce a bottom‐fermented beer. Regarding the buckwheat wort fermentation, a 2‐ to 3‐times lower final acetaldehyde content than in barley is desirable, whereas a relatively high methanol content is not desirable. Barley and buckwheat showed comparable sum concentrations and similar overall profiles of some important aroma compounds. From this perspective, buckwheat appears to be a promising substitute for barley as a brewing raw material. The overall conclusions of our comprehensive study (Parts I–III) are that buckwheat shows adequate brewing properties to substitute for barley in the commercial preparation of a bottom‐fermented gluten‐free beer‐like beverage, and yeast can be repitched at least 11 times. In contrast, quinoa in practical terms shows no substitutional potential for barley in beer; however, it has many nutritious advantages, thus the commercial preparation of a unique, bottom‐fermented gluten‐free ‘non‐beer‐like’ beverage – where the yeast could be repitched six times at most – appears feasible. Copyright © 2015 The Institute of Brewing & Distilling     

Enzymatic and molecular characteristics of Geotrichum candidum strains as a starter culture for malting

Geotrichum candidum yeasts are proposed as a starter culture during malting. They have a double positive effect on the process. They eliminate the fungal pathogenic microflora and improve the technological properties of the finished product, malt. Among published research little or no information can be found considering the biosynthesis of hydrolytic enzymes. Most of the data is related to the production of lipases, proteases and cellulases. This paper examines the enzymology of a number of G. candidum strains. The main focus within the evaluation process was placed on whole cell barley grain cultivation. Hydrolase, which is present in cell wall degradation, was found at a satisfactory level. All tested strains produced both β‐1,3‐glucanases and β‐1,4‐glucanases capable of hydrolysing barley β‐glucan and reducing the amount of this polysaccharide in the wort. Molecular analysis of the tested strains with the use of restriction fragment length polymorphism–polymerase chain reaction and randomly amplified polymorphic DNA confirmed not only the species affiliation, but also the genetic similarity between the tested strains. Copyright © 2014 The Institute of Brewing & Distilling     

Barley Products of Different Fiber Composition Selectively Change Microbiota Composition in Rats

1 Scope

Several dietary fiber properties are suggested to be important for the profiling of the microbiota composition, but those characteristics are rather unclear. Whether different physico‐chemical properties of barley dietary fiber influence the gut microbiota composition is investigated.

2 Methods and results

Seven diets containing equal amounts of dietary fiber from barley malts, brewer's spent grain (BSG), and barley extracts, resulting in varying amounts of β‐glucan, soluble arabinoxylan, and insoluble arabinoxylan in the diets were given to conventional rats. Malts increased microbiota alpha diversity more than BSG and the extracts. The intake of soluble arabinoxylan was related to Akkermansia and propionic acid formation in the cecum of rats, whereas β‐glucan and/or insoluble arabinoxylan were attributed to some potentially butyrate‐producing bacteria (e.g., Lactobacillus, Blautia, and Allobaculum).

3 Conclusion

This study demonstrates that there is a potential to stimulate butyrate‐ and propionate‐producing bacteria in the cecum of rats with malt products of specific fiber properties. Moreover, BSG, a by product from beer production, added to malt can possibly be used to further modulate the microbiota composition, toward a higher butyric acid formation. A complex mixture of fiber as in the malts is of greater importance for microbiota diversity than purer fiber extracts.     

Characterizing and exploiting the rheology of wheat hardness

In the manufacture of cereal products, such as milling of wheat, the mixing of the dough, and baking of cakes and breads, because direct end-product testing is expensive and time consuming, it is popular for industry to utilize appropriate surrogates (representative indirect measurements) to predict various end-product outcomes. For some specific properties, such as milling yield, dough viscoelasticity, or bread loaf volume, the identification and validation of possible surrogates is a major motivating goal in the R&D of cereal science. For example, because end-product quality of many foods made from wheat flour is known to depend principally on the structural and rheological properties of the high molecular weight protein fraction in the flour, the rheology of doughs made from wheat flours is an appropriate and popular choice for the surrogate. Equally important is the end-product testing of the flour milled from a wheat. In this situation, an appropriate surrogate is some measure of the rheology of grain hardness; namely, the deformation and fracturing of the wheat during the milling as a function of the mechanical properties of the various botanical layers within wheat kernels. The current popular measures are particle size index and single kernel characterization system (SKCS) hardness index. However, they are quite indirect and only single-value summaries of the mechanical strengths of the botanical layers. An alternative strategy is the measurement (on an SKCS 4100 device) of a large number of individual crush response profiles which are averaged to produce a representative average crush response profile (aCRP), which can be viewed as an indirect measurement of the strengths of the internal botanical layers. To utilize the rheology of grain hardness as a predictor for the flour that can be milled from various wheats, there is a need to first characterize the information that can be recovered about the mechanical strengths of the internal botanical layers from the morphology of an aCRP. This is the goal and focus of this article. From this perspective, the SKCS 4100 device can be viewed as playing the role of a grain hardness rheometer. Independent motivation identifying a need for an explicit characterization of the rheology of grain hardness is the increasing list of publications exploring and exploiting various features of aCRPs as predictor of key aspects of end-product quality. The results of such publications are briefly reviewed to highlight the growing importance of grain hardness rheology in cereal science. In addition, illustrative examples arising from the current deliberations are included. The SKCS rheological characterization of the strengths of the botanical layers in wheat kernels, as explained in this article, will apply more generally to all cereal grains.