Kappa-Carrageenan Gel Immobilisation of Lager Brewing Yeast

Kappa-carrageenan has been found to be a suitable carrier for yeast immobilisation for continuous fermentation systems due to its unique properties. Brewing lager yeast immobilised in kappa-carrageenan beads was studied both in batch and in a continuously fermenting gas lift bioreactor. The cells were monitored over a six month period of continuous fermentation. Viability (methylene blue), immobilised cell concentration and distribution, and the morphological characteristics of the cells (electron microscopy) were investigated. Viability of cells within the beads was found to decline to less than 50% after six months of continuous wort fermentation and maximum bead population did not exceed 10⁹ cells/mL of gel bead.     

FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR CONTINUOUS BEER FERMENTATION: A REVIEW

Recent fundamental research conducted on immobilised cells with a focus on continuous primary beer fermentation is presented in this review. The knowledge of whole-cell immobilisation, continuous fermentation, yeast biochemistry associated with beer flavour production, and bioreactor engineering design is required to apply immobilised yeast cells for industrial scale beer production. Understanding how immobilisation and continuous bioreactor operation affect yeast cell metabolism and viability will provide the groundwork for optimising beer quality. The latest studies on immobilised cell carriers, viability, vitality, mass transfer characteristics and bioreactor design indicate that an industrial scale immobilised cell system for primary beer fermentation may soon be a reality in the modern brewery .     

NEW DEVELOPMENTS IN THE BREWING INDUSTRY USING IMMOBILISED YEAST CELL BIOREACTOR SYSTEMS*

The use of immobilised yeast cell systems in industry has been extensively reported in the literature. The brewing industry is closely examining immobilisation technology and evaluating its merits. Various immobilisation methods are available to researchers and the nature of the application often dictates the choice of an immobilisation matrix. Industrial scale systems utilising immobilised yeast cells adsorbed to pre-formed carriers have been used for the production of low alcohol beers and for maturation or secondary fermentation of beer. Research relating to the primary fermentation of beer continues and several groups have developed laboratory scale systems. An overview of the respective technologies is provided and several relevant industrial applications cited .     

Effect of Saccharomyces cerevisiae cells immobilisation on mead production

Mead is a traditional alcoholic beverage obtained by the fermentation of diluted honey performed by yeasts. In this work the potential of application of immobilised yeast cells on single-layer Ca-alginate or double-layer alginate–chitosan for mead production was assessed for the first time. The meads produced either with entrapped or free cells were evaluated in terms of quality and aroma profile. The immobilisation procedure had no adverse effect on cell viability, since minor differences were found in fermentation kinetics among the strains and immobilisation systems. The double-layer alginate-chitosan had no advantage compared with the single-layer Ca-alginate, as the number of free cells in the medium, resulting from cell leakage, was similar. Although meads obtained with entrapped yeast cells presented less ethanol and glycerol and more acetic acid, it exhibited larger amounts of volatile compounds. Immobilised cells produced meads with more compounds with fruity characteristics, such as ethyl octanoate and ethyl hexanoate; however the concentrations of undesirable compounds in such meads were also higher. The effect of immobilisation on the aroma profile was important, but the strain contribution was also of major importance. Thus, the sensory analysis of final product gives an important insight on the overall quality.     

Volatile composition of partially fermented wines elaborated from sun dried Pedro Ximénez grapes

In this work, we used a cell immobilisation system consisting of Penicillium chrysogenum fungi (GRAS) bound to the osmotolerant yeast strains Saccharomyces cerevisiae X4 and X5 for the partial fermentation of raisin musts. The resulting wines were compared with others obtained by partial fermentation of musts with free yeasts and with a traditionally produced sweet wine (i.e. without fermentation of the must). The analysis of volatile compounds grouped by aroma series showed the partially fermented musts had a more complex aroma than the traditional wine. Specially prominent among aroma series was that of ripe fruit, followed by the milky and chemical series. The volatiles with the greatest impact on wine aroma as assessed in terms of odour activity were ethyl hexanoate, ethyl octanoate, butyrolactone, isoamyl alcohols, acetaldehyde, ethyl acetate, 2,3-butanediol, acetoin and 2,3-butanedione. A cluster analysis according to the Ward method was performed to assess the similarity between the traditional sweet wine and those obtained by partial fermentation with free and immobilised yeasts revealed small differences between the wines obtained with free and immobilised yeasts, and marked differences between partially fermented and traditionally obtained sweet wine. The wines provided by immobilised yeasts were the most appreciated in the sensory analysis (especially those obtained with X4 yeasts).     

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.     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株.在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失.因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选...     

Bioethanol production from sugar beet molasses and thick juice by free and immobilised Saccharomyces cerevisiae

The aim of this study was the investigation and comparison of the potential of sugar beet molasses and thick juice as raw materials for bioethanol production, as renewable and sustainable energy sources. Ethanol fermentation of a wide range of initial sugar concentrations (100–300 g/L) was performed using either free or immobilised Saccharomyces cerevisiae in calcium alginate beads in the absence of any added nutrients. In general, immobilised cells showed better fermentative performance, enhanced ethanol productivity, stability and cell viability compared with free cells, under the same fermentation conditions. The high concentration of non‐sugar components contained in molasses affected yeast fermentation performance and viability. Maximum ethanol concentration in fermented media of 84.6 and 109.5 g/L were obtained by immobilised cells for initial sugar concentrations of 200 and 250 g/L for molasses and thick juice, respectively. However, the highest ethanol yields of 31.7 L per 100 kg of molasses and 37.6 L per 100 kg of thick juice were obtained by immobilised cells at an initial sugar concentration of 175 g/L. In the high gravity fermentation process, thick juice resulted in a higher ethanol yield per mass of raw material compared with molasses. This study shows the advantage of immobilised yeast for the efficient production of high gravity bioethanol from thick juice, which was a more favourable raw material than molasses. © 2018 The Institute of Brewing & Distilling     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

Industrial wort was fermented with a NewFlo phenotype ale yeast in lab‐scale cylindrical fermenters. The effects of various fermentation parameters and yeast cell wall properties on yeast flocculation were studied during 120 h fermentation. The evaluation of the cell volume during the fermentation revealed a non‐normal distribution (p < 0.05) at most fermentation times. Overall yeast cell size initially decreased during the first 24 h of fermentation then increased during the 24–60 h fermentation period. Cell size subsequently declined until the end of fermentation presumably due to floc settling. While yeast flocculation began after 24 h fermentation, most flocs remained in suspension until 60 h when the average turbulent shear rate caused by CO₂ evolution declined to below 8 s^?1. Both the Helm's flocculence and cell surface hydrophobicity values rapidly increased to high numbers from 24 h onward. Changes in the orthokinetic capture coefficient (α₀) value with fermentation time, measured in fermenting worts, indicated a significant increase (p < 0.001) after 24 h of fermentation. Presumably, this change was due to increases in ethanol and the decline in sugar concentration with time. Although a significant positive correlation (p < 0.05) was observed between zymolectin densities and cell surface areas, the total zymolectin level on yeast cell walls did not change significantly with fermentation time (p > 0.05). Interestingly, no significant difference existed in Helm's flocculation values of suspended and settled yeast cells (p > 0.05). The flocculation rate of LCC125 was readily inhibited by addition of glucose or maltose. Results suggest that fermentable sugar levels and shear force exert major influences on yeast flocculation during beer fermentations.     

Winemaking by barley supported yeast cells

A new biocatalyst was prepared by immobilisation of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole barley grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape must at various temperatures. The biocatalyst retained its operational stability for a long period and it proved capable to produce dry wines of fine clarity even at extremely low temperatures (5 °C). The produced wines were analysed for volatile byproducts by GC and GC–MS. The results showed an increase in the number and amount of esters produced when immobilised cells were used. In addition, an increase in the percentages of esters and a decrease in those of alcohols with the decrease of fermentation temperature were reported.     

Immobilisation and stabilisation of β-galactosidase from Kluyveromyces lactis using a glyoxyl support

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g^?1 of support, resulting in highly active and stable derivatives suitable for industrial processes.     

Characteristics of immobilised β‐glucosidase and its effect on bound volatile compounds in orange juice

In this study, bound volatile compounds were isolated and extracted with Amberlite XAD‐2 resin and then hydrolysed by free or immobilised β‐glucosidase. The released bound volatiles were analysed by GC‐MS. In addition, the optimisation of immobilisation method on sodium alginate and the characteristics of immobilised β‐glucosidase were studied. The results showed that crosslinking‐entrapment was the best method. The optimal conditions of this method were as follows: sodium alginate concentration 3.5%, glutaraldehyde concentration 1%, crosslinking time 3 h, immobilisation time 2 h and CaCl₂ concentration 3%. The optimum temperature for β‐glucosidase (65 °C) was decreased by 10 °C after immobilisation, while the optimum pH values for free and immobilised β‐glucosidase were both at pH 5.0. The K_m values of free and immobilised β‐glucosidase were 14.89 and 0.59 m , respectively. In total, thirteen and six bound volatile compounds were detected in orange juice hydrolysed by free and immobilised β‐glucosidase, including benzenic compounds, terpenic compounds, hydroxy esters, C₁₃‐norisoprenoids and alcohols.     

EFFECT OF ENVIRONMENTAL CONDITIONS ON FLOCCULATION AND IMMOBILISATION OF BREWER'S YEAST DURING PRODUCTION OF ALCOHOL-FREE BEER

A method using immobilised yeasts has been developed and successfully applied for production of alcohol-free beer. The influence of environmental conditions present during alcohol-free beer production on the flocculation and immobilisation of the yeast Saccharomyces cerevisiae var. uvarum was investigated in the present study. In wort, the cells developed flocculation at the end of exponential growth, according to the NewFlo phenotype. In defined medium, the flocculation capacity appeared to be temporary and was lost rapidly during the stationary phase. No increase in cell wall hydrophobicity at the onset of flocculation was observed in either medium. Low growth temperatures increased flocculation capacity approximately four-fold, compared to growth at high temperatures. The optimum temperature for flocculation was at 25°C with cells grown at low or high temperature . A novel method using carboxyfluorescein-stained cells was developed to analyse the initial adhesion of cells to carrier. This method also allowed rapid analysis of the effects of immobilisation to DEAE-cellulose carrier during alcohol-free beer production process. It appeared that a high flocculation capacity stimulated adhesion to the DEAE-carrier .     

Growth characteristics of agrowaste‐immobilised lactobacilli in soymilk during refrigerated storage

We aimed at evaluating agricultural wastes as solid supports for the immobilisation of lactobacilli in a liquid media, namely soymilk. Lactobacillus acidophilus FTDC 1331, L. acidophilus FTDC 2631, L. acidophilus FTDC 2333, L. acidophilus FTDC 1733 and L. bulgaricus FTCC 0411 were immobilised on solid supports produced from durian (Durio zibethinus), cempedak (Artocarpus champeden) and mangosteen (Garcinia mangostana). The immobilised cells were inoculated into soymilk and stored at 4 °C over 168 h. Soymilk inoculated with non‐immobilised cells was used as the control. Immobilised cells showed higher survivability over 168 h compared to the control, accompanied by higher reduction of simple sugars and oligosaccharides in soymilk. Higher growth and higher utilisation of substrates also led to the higher production of lactic and acetic acids, which lowered the pH in soymilk compared to the control. Our results illustrated that agrowastes could be used as immobilisers to enhance the growth of lactobacilli in a liquid medium.     

Immobilised yeast grape must deacidification in a recycle fixed bed reactor

Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium‐alginate double‐layer beads (ProMalic^®) was studied in Erlenmeyer flasks and in a total recycle fixed‐bed reactor operating in batch mode. The reaction is pseudo‐first order with respect to l ‐malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol^?1) in comparison with the free cells (126 ± 19 kJ mol^?1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale‐up of a recycle reactor to process large volumes of grape must.     

Application of novel electrospun nanofibrous membranes with different modifications for immobilising glutamine synthetase

BACKGROUND: Theanine synthesis is of interest because of the physiological and pharmacological benefits of theanine. Glutamine synthetase (GS, EC 6.3.1.2) from Pseudomonas taetrolens can be used for theanine biosynthesis. In this study, GS was immobilised in polyvinyl alcohol electrospun nanofibres, and the properties of immobilised GS with different solidifying modifications were studied. RESULTS: Electrospinning is a good method for enzyme immobilisation. When carbon nanotubes were used as an adsorbent, the activity retention of immobilised GS was 92.3%. With glutaraldehyde as a crosslinker, the enzyme activity retention was only 50.7%; however, by adding collagen or peptide to the electrospinning solution with glutaraldehyde, the activity retention could be improved to 85.9 or 59.6% respectively. Scanning electron micrographs showed that the modifiers induced morphological changes in the nanofibres. The optimum pH and temperature of immobilised GS also changed. In addition, the reusability and storage stability of immobilised GS with glutaraldehyde were much better than those of immobilised GS with carbon nanotubes. CONCLUSION: A novel method has been established in this study for the immobilisation of GS in electrospun nanofibrous membranes. Carbon nanotubes and glutaraldehyde enhanced the immobilisation, while collagen or peptide inclusion could recover the activity loss caused by glutaraldehyde. The obtained immobilised enzyme achieved high reusability and storage stability. Copyright © 2012 Society of Chemical Industry     

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

Peanut α‐galactosidase was immobilised in calcium alginate beads and used to hydrolyse the flatulence‐causing oligosaccharides, raffinose and stachyose, in soya milk in batch and in packed bed reactor with recycle. The immobilised enzyme exhibited a slightly lower activity than the free enzyme. The activity yield of immobilised α‐galactosidase was 75.1% and the immobilisation yield was 82.6%. Batch hydrolysis using immobilised enzyme at 55 °C resulted in 96% reduction in the oligosaccharides after 12 h. For the continuous process, a packed bed reactor with recycle was used. More than 98% of the oligosaccharides were hydrolysed after 6 h of reaction at 55 °C. The immobilised enzyme also proved to be stable up to three repeated hydrolysis reactions.     

Influence of yeast strain,immobilisation and ageing time on the changes of free amino acids and amino acids in peptides in bottle‐fermented sparkling wines obtained from Vitis vinifera cv. Emir

This paper reports the influence of yeast strain, immobilisation and ageing time on the free amino acids and amino acids in peptides of sparkling wine made by the traditional method from the Emir grape variety over a period of 365 days in vintages 2004 and 2005. Four sparkling wines were obtained from the same base wine using yeast strains (both immobilised and free Saccharomyces bayanus and Saccharomyces oviformis). The analysis of variance and cluster revealed that there were significant differences between free amino acids and amino acids in peptides due to ageing time. However, the yeast strain affected most of the free amino acids and amino acids in peptides in only the 2004 vintage. The total amount of free amino acids was higher in the wine made with the S. oviformis compared to S. bayanus. Furthermore, there were no significant differences between the use of immobilised and free yeast in cases of free amino acids and amino acids in peptides.     

Effect of additives on the rehydration of Saccharomyces cerevisiae wine strains in active dry form: influence on viability and performance in the early fermentation phase

The effect of five additives on the rehydration process of three commercial Saccharomyces cerevisiae wine strains, in the form of active dry yeast, was tested using the approach of design of experiments. The response to additives was monitored by observing the viability, measured using fluorescence methodology at the end of the rehydration experiments, and fermentative performance, focusing attention on the first phase of fermentation, assessed based on ethanol production and the total number of cells after 48 h from the time of inoculation in synthetic must. The results showed a correlation between viability and the presence of magnesium in the rehydration medium. However, improvement in viability was not associated with better fermentation performance, which was not affected or in some cases significantly reduced. Among the additives examined, only rehydration with inactive dry yeast and ammonium showed a positive effect on the subsequent fermentations, but not for all of the strains tested. In general, considering all of the additives tested, no relationship was found between viability at the end of rehydration and fermentation performance. These findings suggest that the viability of the rehydrated yeast cells may not be a good index of fitness in the subsequent fermentation. Copyright © 2014 The Institute of Brewing & Distilling