Determination of Antifungal Effect of Edible Coatings Containing Williopsis saturnus var. saturnus Against Yeast and Mold Growth on Kashar Cheese

One of the most important problems of Kashar cheese producers is mold and yeast spoilage during storage. Williopsis saturnus var. saturnus killer yeast has been reported to have an antagonistic effect on mold and yeast reproduction. In this study, the antifungal effect of a whey protein concentrate (WPC) coating containing W. saturnus was investigated in Kashar cheese. WPC with or without W. saturnus (7 log CFU/g) or W. saturnus without coating was applied on the surface of Kashar cheese samples and stored at 4 °C for 56 days. Microbiological, chemical, physical and sensory properties of cheese samples were assessed. After 56 days of storage, the numbers of lactic acid bacteria were not affected by WPC containing W. saturnus; however, the population of W. saturnus increased by 1 log CFU/g. Application of W. saturnus reduced the growth of other yeasts and molds (P < 0.05). Chemical, physical and sensory properties of all coated cheeses remained unchanged. In a conclusion, use of WPC coatings containing W. saturnus can potentially minimize mold and yeast spoilage of cheese during storage.     

Modulation of grape wine flavor via the sequential inoculation of Williopsis saturnus and Saccharomyces cerevisiae

The effects of the inoculum ratio of Williopsis saturnus var. saturnus NCYC22 and Saccharomyces cerevisiae var. bayanus EC-1118 at 1:200 and 1:800 on the chemical and volatile compositions of grape wine were studied in sequential fermentation. The grape juice was first inoculated with Williopsis (W.) saturnus for 9 d; thereafter, Saccharomyces (S.) cerevisiae was inoculated to continue the fermentation until d 19. The cell population of W. saturnus disappeared by d 13, with S. cerevisiae dominating until the end of the fermentation in both inoculum ratios. The changes in yeast count, pH, total soluble solids, sugars, organic acids, and amino acids were similar between the two inoculum ratios. A range of volatile compounds was formed, including alcohols, esters, fatty acids, aldehydes, and terpenes. There were significant differences between both inoculum ratios for medium-chain fatty acids (C8, C10, and C12), ethyl esters of fatty acids of C6, C10, C12, and C14 as well as isoamyl octanoate, while other volatiles were statistically the same.     

Enhanced production of isoamyl acetate from beet molasses with addition of fusel oil by Williopsis saturnus var. saturnus

Fusel oil which contains high level of amyl alcohols (approximately 45–55%) is a by-product obtained from the distillation of alcohol made by fermentation of molasses. Williopsis saturnus is a yeast which is able to convert isoamyl alcohol into isoamyl acetate. The aim of this study was to increase the formation of isoamyl acetate by the addition of fusel oil at the ratios of 1%, 2% and 3% (v/v) to molasses based fermentation medium using W. saturnus. It was found out that bioconversion of added fusel oil into isoamyl acetate was possible and an addition of 1% fusel oil led to an increase in isoamyl acetate concentration from 118 to 354 mg/L.     

Growth and fermentation kinetics of a mixed culture of Saccharomyces cerevisiae var. bayanus and Williopsis saturnus var. saturnus at different ratios in longan juice (Dimocarpus longan Lour.)

Cofermentation of longan juice by mixed cultures of Saccharomyces cerevisiae var. bayanus EC‐1118 and Williopsis saturnus var. saturnus CBS254 at two inoculation ratios (EC‐1118:CBS254 = 1:100 and 1:1000 cfu mL^?1) was performed to ascertain their impact on longan wine aroma compound formation. The results showed improved aroma compound profiles in the longan wine fermented with mixed yeasts in comparison with the longan wines fermented with single yeasts in terms of increased production of acetate esters, fatty acid ethyl esters, alcohols and carboxylic acids. The impact of cofermentation on longan wine aroma formation was affected by the ratio of S. cerevisiae EC‐1118 to W. saturnus CBS254 with 1:100 cfu mL^?1 being more effective. This research suggests that the inoculation ratio of mixed yeasts may be used as an effective means of manipulating longan wine aroma.     

Influence of carbon and nitrogen source on production of volatile fragrance and flavour metabolites by the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography–mass spectrometry (GC–MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2‐phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2‐phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non‐Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production. Copyright © 2014 John Wiley & Sons, Ltd.     

Isolation of a Copper‐resistant Sake Yeast Mutant with Improved Flavour Compound Production

The sake (traditional Japanese alcoholic beverage) yeast mutant A1 was previously isolated as a strain resistant to an isoprenoid analog. This strain is used for industrial sake brewing because of its increased production of isoamyl acetate. In this study, a physiological event was identified which was closely related to the elevation of alcohol acetyltransferase (AATase) activity in strain A1. This finding was applied for the isolation of another mutant with an improved capacity for flavour compound production. Strain A1 revealed an additional phenotype showing resistance to Cu²⁺, as seen from its growth and isoamyl acetate production, even in a medium with the copper ion at 6 mM. Mutant strains were successfully isolated with increased isoamyl acetate production capacity from sake yeast strain 2NF on the basis of a Cu²⁺‐resistant phenotype at a high yield. Among them, strain Cu7 was characterized by its ability to produce isoamyl acetate at the highest concentration under condition where isoamyl alcohol (its precursor) was accumulated to the lowest extent. Such a phenotype of strain Cu7 is applicable for the practical production of an alcoholic beverage of excellent quality in terms of flavour.     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

Antifungal,Mechanical, and Physical Properties of Edible Film Containing Williopsis saturnus var. saturnus Antagonistic Yeast

The molding of food products causing health risks is a main problem in the food industry. In this study, as an alternative solution for preventing mold growth, an antifungal edible film was developed by incorporating Williopsis saturnus var. saturnus (0; 3; 7; and 9 logs CFU/cm²) into whey protein concentrate (WPC) based films. Antifungal properties of the films against Penicilium expansum and Aspergillus niger were analyzed using the disc diffusion method. Physical (barrier, solubility, color), mechanical (tensile strength and percent elongation) properties of the films as well as the survival of W. saturnus in the film were assessed during 28 days of storage at 23 °C. According to the results, the viability of W. saturnus (7 and 9 logs CFU/cm²) in WPC films stored for 28 days under vacuum or non‐vacuum decreased to 36% and 60%, respectively. In addition, films containing W. saturnus decreased the viability of P. expansum and A. niger by 29% and 19%, respectively. Adding yeast did not change the tensile strength (P > 0.05), but significantly decreased % elongation and increased water vapor and oxygen permeability and water solubility (P < 0.05). In conclusion, this study showed that the developed films may be useful for inhibiting mold growth on foods.     

Effects of Pure and Mixed-Cultures of Saccharomyces cerevisiae and Williopsis saturnus on the Volatile Profiles of Grape Wine

Non-Saccharomyces yeasts are being recognised for their ability to produce wines with unique character. This study assessed the production of volatiles by co-inoculating Saccharomyces cerevisiae and Williopsis saturnus. Laboratory-scale fermentations were carried out using pure and mixed-cultures of S. cerevisiae and W. saturnus at a ratio of 1:1000. The mixed-culture fermentation was dominated by S. cerevisiae, while W. saturnus had an early growth arrest. Changes of oenological parameters and volatiles were similar in both the mixed- and the S. cerevisiae monocultures. A range of volatiles was formed with alcohols and esters constituting the majority of volatiles produced. Volatiles initially present in the grape juice, particularly (E)-2-hexen-1-ol, 1-hexanol, trans-2-hexenal and n-hexanal, were metabolized. Wines produced using mixed-cultures closely resembled those fermented by the S. cerevisiae monoculture but acquired minor flavor characteristics from the initial presence of W. saturnus. These findings suggest that the ratio of S. cerevisiae and W. saturnus is critical to the survival of the non-Saccharomyces yeast and the impact on the perceivable characteristics of the resultant quality and flavor of wines.     

Contribution by Saccharomyces cerevisiae Yeasts to Fermentation and Flavour Compounds in Wines from cv. Kalecik karasi Grape

The effect of indigenous and commercial S. cerevisiae yeasts on fermentation and flavour compounds of wines was examined in pasteurised grape juice. The flavour compounds were analysed and identified by GC‐FID and GC‐MS, respectively and in general, the amounts of these volatiles were increased by the use of both indigenous and commercial yeasts. The levels of isoamyl alcohol, isoamyl acetate, ethyl octanoate and ethyl deconoate exceeded flavour thresholds. All grape juices were fermented to dryness. Selected yeasts produced higher ethanol concentrations compared to spontaneous fermentations.     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

Evolution of volatile compounds in papaya wine fermented with three Williopsis saturnus yeasts

The changes in volatile compounds during papaya juice fermentation with three Williopsis saturnus yeasts were investigated in this study. Time‐course papaya juice fermentations were carried out using three Williopsis saturnus yeasts: W. saturnus var. mrakii NCYC2251, W. saturnus var. saturnus NCYC22 and W. saturnus var. sargentensis NCYC2727. Changes in yeast cell population, Brix and pH were similar among the three yeasts, which preferentially utilised glucose over fructose while partially degrading l ‐malic acid. A range of volatile compounds were produced during fermentation including fatty acids, alcohols and esters with esters being the most abundant volatile compounds produced. Benzyl isothiocyanate, butyric acid, 2‐ethylhexanol, benzaldehyde and β‐damascenone present in the papaya juice were metabolised to trace levels during fermentation. There were significant variations among the three yeasts in their ability to produce and metabolise volatile compounds during fermentation. The study suggests that papaya juice fermentation with W. saturnus yeasts is able to result in the formation of a more complex aroma compounds.     

Effects of sequentially inoculated Williopsis saturnus and Saccharomyces cerevisiae on volatile profiles of papaya wine

The effects of sequential inoculation of yeasts Williopsis saturnus var. mrakii NCYC2251 and Saccharomyces cerevisiae var. bayanus R2 on the volatile profiles of papaya wine were investigated at an inoculum ratio of 1000 (W. saturnus) to 1 (S. cerevisiae). Inoculation of S. cerevisiae after seven days' fermentation with W. saturnus produced papaya wine with more acetate esters and fruitiness than the control (simultaneous inoculation). However, inoculation of W. saturnus after two days' fermentation with S. cerevisiae resulted in most of the volatile composition being comparable to the control, except for the enhanced amount of ethyl esters. The first inoculated yeast dominated the fermentation. The study suggests that sequential inoculation of non-Saccharomyces and Saccharomyces yeasts at a certain inoculum ratio may be a valuable tool to manipulate yeast succession and to modulate the volatile profiles and organoleptic properties of papaya wine.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Using spray-dried sugar beet molasses in ice cream as a novel bulking agent

In this study, it is aimed to investigate and evaluate the use of molasses which is a by-product of sugar production as a novel bulking agent in ice cream as a sugar replacer. Sugar beet molasses (75%) and 12 DE maltodextrin (25%) were converted into powder form by spray drying. Spray-dried sugar beet molasses (SDSM) was then used as a substitute of sugar in different ratios (0:100, 25:75, 50:50, 75:25 and 100:0) in the ice cream products. The increased amount of SDSM decreased the overrun, L*, whiteness index (WI) and melting behaviours and increased a*, b*, total phenolic content, consistency and viscosity. Meanwhile, thermal properties have not been affected by the use of SDSM (P < 0.05). The sensory findings have been particularly interesting especially when replacing above 50% sugar with SDSM, aroma, flavour and general acceptability decrease. According to the results of this study, substituting 25% of total sugar with SDSM as a bulking agent can decrease cost of the product and improve total phenolic content and some quality parameters without compromising sensorial properties.     

Production of alcohol‐free beer with elevated amounts of flavouring compounds using lager yeast mutants

Arrested or limited fermentation process is a widespread method used for production of alcohol‐free beer (AFB). However, it usually leads to worty off‐flavour and a lack of pleasant fruity flavour/aroma. The aim of this study was to isolate spontaneous mutants of Saccharomyces pastorianus strain 2 resistant to 5,5,5‐trifluoro‐dl ‐leucine, since the resistance is related to overproduction of flavour active isoamyl alcohol (IAAL) and isoamyl acetate (IAAC). The mutants were the subject of selection during series of screening tests aimed at selecting the best producer of target compounds (IAAL and IAAC). Subsequently, the overproduction stability of the selected mutant strain was verified in four consecutive fermentations of AFBs. A higher pitching rate and increased fermentation temperature did not result in an overall improvement in the production of the target compounds. Sensory analysis proved the flavouring effect of the spontaneous lager yeast mutant on the AFB produced by arrested fermentation. Copyright © 2013 The Institute of Brewing & Distilling     

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

利用廉价原料生产聚谷氨酸的研究

目的 降低聚谷氨酸的生产成本.方法 从聚谷氨酸发酵所需的氮源和碳源出发,分别研究了小麦水解蛋白、豆饼粉、豆粕粉、玉米蛋白、玉米浆等有机氮源和甘蔗糖蜜、甜菜糖蜜、葡萄糖蜜、玉米糖蜜等碳源对聚谷氨酸发酵产量的影响,在此基础上通过单因素实验优化了玉米浆、硫酸铵、甜菜蜜、谷氨酸钠的添加量,最后通过发酵罐发酵比较了优化后的工艺与...     

CARBON SOURCES AND THEIR EFFECT ON GROWTH, ACETIC ACID AND ETHANOL PRODUCTION BY BRETTANOMYCES BRUXELLENSIS IN BATCH CULTURE

The influence of available low‐cost carbohydrates as carbon sources on Brettanomyces bruxellensis growth, acetic acid and ethanol production was studied in order to ascertain the viability of this yeast to eventually become an industrial acetic acid producer. Six different raw materials were included as carbon sources (glucose, sugarcane molasses, refined cane sugar, pineapple, sugarcane and beet juices). B. bruxellensis develops in a complex culture medium like plant juices and sugarcane molasses better than in a medium with a simple carbohydrate such as glucose. The maximum acid acetic yield (0.24 g/g) and productivity (0.14 g/L/h) were attained in tests carried out with sugarcane molasses containing 60 g/L sucrose. The strain produced low levels of ethanol in a refined sugarcane medium, but was able to produce a substantial quantity of acetic acid (13 g/L).     

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.