Immobilization techniques for beverage production using yeast cell systems: challenges, types, and future perspectives - a mini review

Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventional methods.Research highlights that immobilized yeast cell systems have several benefits as compared to free yeast cells.The immobilized yeast cell systems improve fermentation rates,especially when paired with continuous fermentation and appropriate immobilization techniques.Understanding various immobilization techniques,continuous fermentation processes,yeast metabolic activity related to beverage flavor production,and bioreactor designs is vital for optimizing the use of immobilized yeast cells systems on industrial scale.This review provides an overview of recent basic research on immobilized yeast cell systems,with a focus on continuous beverage fermentation.In this study,different reactor configurations and immobilization techniques are explored.The study focus on the impacts of immobilization on the yeast cells,and discuss the recent advancements in these techniques.The review concludes with a discussion on the practical applications of immobilized yeast cells and continuous fermentation in beverage production.

Cell Toxicity Effect of Cadmium on Yeast Cells by Detecting NADH Autofluorescence

The cytotoxic effect of cadmium is studied by detecting intracellular nicotinamide adenine dinucleotidea（NADH） autofluorescence in this work. NADH autofluorescence in processes of cadmium-induced apoptosis, necrosis and reversible injury are recorded timely. The relativity between time course of NADH autofluorescence and cadmium toxicity is established. The cell toxicity effect of Cadmium on yeast cells is studied by detecting the time courses of intracellular reduced NADH autofluorescence in this work. The relativity between time courses of NADH autofluorescence and Cadmium toxicity is established.

Mediated Electrochemical Measurements of Intracellular Catabolic Activities of Yeast Cells

Coupling with the dual mediator system menadione/ferricyanide, microelectrode voltammetric measurements were undertaken to detect the ferrocyanide accumulations arising from the mediated reduction of ferricyanide by yeast cells. The results indicate that the dual mediator system menadione/ferricyanide could be used as a probe to detect cellular catabolic activities in yeast cells and the electrochemical response has a positive relationship with the specific growth rate of yeast cells.

Effects of Dietary Yeast Cell Wall Polysaccharides Supplementation to Late Gestation and Lactation Diets on Reproductive Performance and Blood Profiles of Sows and Their Offspring

This study was conducted to determine the effects of yeast cell wall polysaccharides（YCWP） on reproductive performance, serum profiles and milk composition during late gestation and lactation. A total of 96 sows（Landrace × Yorkshire） were blocked on a basis of breed, parity（2-3）and their respective d 85 of gestation, and then randomly allotted to the following three dietary treatments： CON（basal diet, treatment 1）, YCWP1（CON＋0.2% YCWP, treatment 2） and YCWP2（CON＋0.4% YCWP, treatment 3）. The experiment lasted from d 85 of gestation to weaning（d 21 of lactation）. Sows fed YCWP diets had lower backfat thickness reduction from postpartum to weaning（ P〈0.05）. The supplementation of YCWP tended to increase litter weight at weaning（P=0.08） compared with CON. The non-lipid and total milk solids in colostrum was improved by YCWP2（P〈0.05）.No differences were observed in milk composition among dietary treatments（ P〉0.05）. The supplementation of YCWP decreased（P〈0.05） the levels of alanine amino transferase（ALT）, aspartate amino transferase（AST）, while it did not affect（ P〉0.05） alkaline phosphatase（ALP）, total protein（TP）,albumin（ALB）, globulin（GLB）, glucose（GLU）, total cholesterol（TC）, triglyceride（TG） or immunoglobulin G（IgG） in the sow serum. The serum IgG of piglets was increased by the YCWP. The levels of serum ALT and AST were reduced（P〈0.05） for suckling piglets in YCWP treatments. Taken together, the results of current studies might indicate beneficial effects of YCWP supplementation to late gestation and lactation diets on the sow productivity by improving litter weight at weaning, reducing backfat loss, and increasing serum IgG concentrations of piglets.

STUDY ON IMMOBILIZED YEAST CELLS WITH HRDROPHILIC POLYMER CARRIER BY RADIATION - INDUCED COPOLYMERIZATION

In this paper, various kinds of monomers 2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl acrylate (HEA), hydroxypropyl methacrylate (HPMA) and methoxy polyethylene glycol methylacrylate (M-23G) were eopolymerized by radiation technique at low temperature (-78℃) and several kinds of copolymer carriers were obtained. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves on these carriers. The etharol productivity of immobilized yeast cells with these carriers was related to the monomer composition and water content of copolymer carriers and the optimum monomer composition was 20%:10% in poly (HEA-M23G). In this case, the ethanol productivity of immobilized yeast cells was 26 mg /( ml·h), which was 4 times as high as that of free cells. In this study, the effect of adding of crosslinking reagent (4G) in copolymer on activity of yeast cells immobilized with the carriers were also studied. It was found that the effect of adding crosslinking reagent (4G) in lower monomer composition of poly(HEA-M23G) on the ethanol productivity of immobilized cells was better than that in higher one in this work.

Inactivation cross section of yeast cells irradiated byheavy ions

Inactivation cross sections for haploid yeast cell strain 211a have beencalculated as 1-hit detector based on the track theory in an extended target mode anda numerical calculation of radial dose distribution. In the calculations, characteristicdose Do is a fitted parameter which is obtained to be 42 Gy, and "radius" of hypothetical target ac is chosen to be 0.spin which is about the size of nucleus of yeastcells for obtaining an overall agreement with experimental cross sections. The resultsof the calculations are in agreement with the experimental data in high LET (linearenergy transfer) including the thindown region.

Micro-environmentally Restricted Yeast Cell Growth within Ca-alginate Microbeads

Micro-environmental restriction effects to yeast cell growth obtained within Ca-alginate microbeads are considered. It is complex phenomenon influenced by: (1) relaxation of expanded polymer network around the cellular clusters, (2) forces generated by cell growth inside the beads and (3) interactions between solvent, network parts and cells. The resulting effects are measured experimentally by estimating volume of microbeads and yeast cell concentration as function of time of cultivation. Comparative analysis of dynamics of cell growth and increase of microbead volume through four regimes indicates that reversible and irreversible local structural changes of Ca-alginate hydrogel induces micro-environmental restrictions to cell growth. The mechanism of restrictions includes both mechanical and electrostatic effects.

Impacts of low level aflatoxin in feed and the use of modified yeast cell wall extract on growth and health of nursery pigs

This study was to investigate the effect of corn naturally contaminated with aflatoxins(AF)under the regulatory level on the growth performance and health of nursery pigs,and the efficiency of yeast cell wall based feed additive(YC)mainly composed ofβ-glucans and mannan oligosaccharide(MOS)(Integral A+,Alltech,Lexington,KY)in prevention of mycotoxicosis.Pigs(60 barrows and 60 gilts at 6.02 ± 0.83kg BW)were randomly allotted to 4 treatments in a randomized complete block design based on a 2×2 factorial arrangement with 10 pens(5 barrow and 5 gilt pens)per treatment and 3 pigs per pen.Pigs were fed experimental diets for 5 wk.First factor was AF(0 or 20 mg/kg in feed)and the second factor was YC(0 or 2 g/kg in feed).Feed intake and body weight were measured weekly,and blood samples were used to measure blood cell counts,immunoglobulin G(Ig G),tumor necrosis factor-a(TNF-a),oxidative damage status,and serological evaluation related to liver health.Aflatoxin decreased(P<0.05)the number of platelet count(247.4 to 193.5×10~3/μL),and it also tended to increase the level of albumin(P=0.055,3.46 to 3.63 g/d L),albumin:globulin ratio(P=0.050,2.09 to 2.37),and Ca(P=0.080,10.79 to 10.97 mg/d L).Yeast cell wall based feed additive increased(P < 0.05)ADG(493 to 524 g/d),and ADFI(796 to 846 g/d)of pigs whereas G:F was not affected,and it also tended to increase(P=0.055)albumin level(3.46 to 3.63 g/d L).Interactions(P < 0.05)on hemoglobin,hematocrit,and platelet count indicated that YC further increased their levels when pigs were eating AF contaminated feed.Interactions(P < 0.05)on urea nitrogen and blood urea N to creatinine ratio indicated that YC further decreased their levels when feed were contaminated with AF.In conclusion,low level of 20 mg AF/kg under the regulatory level had minor effects on hematology without affecting growth performance,however the supplementation of 2 g/kg YC as a source ofβ-glucans and MOS in feed can improve feed intake and therefore the growth of pigs.

Effects of yeast cell wall on growth performance, immune responses and intestinal short chain fatty acid concentrations of broilers in an experimental necrotic enteritis model

Subclinical necrotic enteritis(NE) causes devastating economic losses in the broiler chicken industry,especially in birds raised free of in-feed antibiotics. Prebiotics are potential alternatives to in-feed antibiotics. Yeast cell wall extract(YCW) derived from Saccharomyces cerevisiae is a prebiotic with known immune modulating effects. This study examined the effects of YCW and antibiotics(AB) during subclinical NE on broiler growth performance, intestinal lesions, humoral immune response and gut microflora metabolites. The study employed a 2 × 3 factorial arrangement of treatments. Factors were:NE challenge(yes or no) and feed additive(control, AB, or YCW). Each treatment was replicated in 8 floor pens with 15 birds per pen. Challenged birds had higher feed conversion ratio(FCR) than unchallenged birds on d 35(P < 0.05). Dietary inclusion of AB decreased FCR regardless of challenge(P < 0.05) on d 24 and 35. Inclusion of YCW reduced serum interleukin-1(IL-1) concentration in NE challenged birds(P < 0.01) and increased immunoglobulin(Ig) G(P < 0.05) and Ig M(P < 0.05) levels compared to other dietary treatments regardless of challenge. Yeast cell wall extract increased formic acid concentration in cecal contents during challenge and increased butyric acid concentration in unchallenged birds on d 16.This study indicates YCW suppressed inflammatory response, promoted generation of immunoglobulin and increased short chain fatty acid production suggesting potential benefits to bird health.

Dietary supplementation of yeast cell wall improves the gastrointestinal development of weaned calves

The purpose of this study was to investigate the potential benefits of yeast cell wall(YCW)on the gastrointestinal development of weaned calves.Twenty healthy Holstein male calves(BW=92±8.29 kg and 60±5 d of age)were randomly allocated into 2 groups:CON with no YCW,and YCW(accounted for0.16%of the basal diet).The dietary concentrate-to-roughage ratio was 40:60.All the calves were fed regularly twice a day at 09:00 and 16:00 and had free access to water.The experiment lasted for 60 d.The results showed that calves fed YCW showed higher(P<0.05)length,width,and surface area of papillae in the ventral sac of the rumen as compared to CON.For the dorsal sac of the rumen,the muscularis thickness was thicker(P<0.05)in the YCW group when compared with CON group.The villus height of YCW calves was higher(P<0.05)than that of CON in the ileum.Calves supplemented with YCW also showed a higher(P<0.05)villus height-to-crypt depth ratio in the ileum.The YCW calves exhibited a greater(P<0.05)thickness of the wall in the duodenum and jejunum.Calves supplemented with YCW improved(P<0.05)the claudin 1 mRNA expression in the ileum and occludin mRNA expression in the jejunum and ileum.The YCW increased(P<0.05)the contents of secretory immunoglobulin A in the jejunum and ileum of calves.In conclusion,dietary supplementation with YCW could improve the gastrointestinal development of weaned calves.

Use of dry yeast cells as a cheap nitrogen source for lactic acid production by thermophilic Bacillus coagulans WCPIO-4

Dry yeast cells （DYC） were used as a cheap nitrogen source to replace expensive yeast extract （YE） for L-lactic acid production by thermophilic Bacillus coagulans. Cassava starch （200 g. L1） was converted to L-lactic acid by simultaneous saccharification and fermentation using Bacillus coagulans WCP10-4 at 50℃ in the presence of 20g·L-1 of DYC, giving 148.1 g·L-1 of L- lactic acid at 27 h with a productivity of 5.5 g·L-1·h-1 and a yield of 92%. In contrast, 154.4 g·L-1 of lactic acid was produced at 24 h with a productivity of 6.4 g·L-1. hl and a yield of 96% when equal amount of YE was used under the same conditions. Use of pre-autolyzed DYC at 50℃ for overnight slightly improved the lactic acid titer （154.5g·L-1） and productivity （7.7g·L-1·h-1） but gave the same yield （96%）.

Sequential co-immobilization of β-glucosidase and yeast cells on single polymer support for bioethanol production

Co-immobilization of enzymes and microorganism is an effective way to enable cells to use nonmetabolizable substrates and accelerate reaction rate of overall process. Herein, a facile strategy to separately co-immobilize β-glucosidase(BG) and yeast cells on non-woven fabrics was developed. The BG was firstly in situ entrapped into poly(ethylene glycol)(PEG) network grafted on non-woven fabrics by visible light induced living/controlled graft polymerization. Then re-graft polymerization was performed on the as-formed BG loaded layer by taking advantage of living-grafting polymerization on its surface to in situ encapsulate yeast cells into the second PEG network layer. This layered structure of co-immobilization avoided possible interference between enzyme and cells. Viability assay of yeast cells demonstrated that most of cells were viable after immobilization. While immobilized BG showed decreased V_(max) compared to free BG, indicating that entrapping BG into inner PEG network layer restricted its accessibility with substrates. This co-immobilization sheet could successfully convert cellobiose to ethanol and a maximum of 98.6% bioethanol yield can be obtained after 48 h of simultaneous saccharification and fermentation(SSF). The co-immobilization sheet showed excellent reusability and could still reach more than 60% of original ethanol yield after reusing for 7 batches. Compared with the mixed co-immobilization, the sequential layered immobilization in this system showed better stability and higher ethanol yield.

酵母复合微生态制剂对鲤鱼生长性能、免疫指标及抗氧化能力的影响

本试验旨在研究饲料中添加酵母复合微生态制剂对鲤鱼生长性能、免疫指标及抗氧化能力的影响,并确定其适宜的添加量。选用初始体重为(35±0.5)g的鲤鱼540尾,随机分为6组,每组3个重复,每个重复30尾鱼,预饲14 d。以基础日粮为CK组,试验组分别饲喂含0.1‰、0.5‰、1‰、2‰、3‰酵母复合微生态制剂饲料,饲养56 d后采样分析。结果表明:(1)与CK组相比,饲料中添加酵母复合微生态制剂可显著提高鲤鱼的生长性能,2‰、3‰组增重率(WGR)增加了23.01%、24.15%(P<0.05),特定生长率(SGR)增加了13.79%、14.37%(P<0.05),饲料系数(FCR)降低了18.09%、18.59%(P<0.05)。(2)与CK组相比,饲料中添加酵母复合微生态制剂可显著提高鲤鱼的免疫指标,2‰、3‰组溶菌酶(LSZ)提高了47.9%、48.74%(P<0.05),免疫球蛋白M(IgM)提高了48.31%、49.44%(P<0.05),补体C3(C3)提高了39.61%、39.61%(P<0.05),碱性磷酸酶(AKP)提高了17.05%、17.44%(P<0.05),总蛋白(TP)提高了31.03%、30.38%(P<0.05)。(3)与CK组相比,饲料中添加酵母复合微生态制剂可显著提高鲤鱼的抗氧化能力,2‰、3‰组总抗氧化能力(T-AOC)提高了41.69%、41.36%(P<0.05),超氧化物歧化酶(SOD)提高了37.63%、45.71%(P<0.05),过氧化氢酶(CAT)提高了41.69%、41.36%(P<0.05),丙二醛(MDA)降低了22.71%、20.92%(P<0.05)。由此得出,饲料中添加2‰、3‰酵母复合微生态制剂能够显著提高鲤鱼的生长性能、血清免疫指标及肝脏抗氧化能力。基于2‰、3‰添加量组各指标差异不显著,建议酵母复合微生态制剂的最佳添加剂量为2‰。

酵母细胞壁来源、作用机理及在动物养殖中的应用

酵母细胞壁具有吸附病原微生物、调整有益菌落和调节免疫功能等作用,其作为酵母类生物饲料原料的重要组成部分,已经被广泛应用于畜禽养殖的各个环节。本文就酵母细胞壁来源、组成、作用机理及在动物养殖中的应用进行综述,以期为酵母细胞壁在实际生产中的合理应用提供理论参考。

非酿酒酵母与酿酒酵母在米酒混菌发酵中的相互作用机制分析

该研究以传统米酒酿造中常见的两种典型非酿酒酵母,假丝酵母(Candida glabrata,Ca)和扣囊复膜酵母(Saccharomycopsis fibuligera,Sf)为研究对象,利用同时接种和顺序接种两种发酵方式分别与酿酒酵母(Saccharomyces cerevisiae,Sc)进行米酒混菌发酵,探究非酿酒酵母和酿酒酵母间的相互影响及相关作用机制。研究结果表明,虽然2种混菌发酵方式表现出明显不同的发酵特性,但在混菌发酵过程中,当Sc生物量达到一定水平(约107 CFU/mL)后,2株非酿酒酵母的生长均受到显著的抑制,生物量快速下降,而Sc生物量基本未受非酿酒酵母的影响。细胞非接触发酵实验结果表明,一定浓度的Sc与非酿酒酵母的细胞-细胞接触是产生抑制的主要原因。Sc发酵代谢物以及酒精和营养物质含量对非酿酒酵母的生长影响不大。

酵母细胞催化合成18α-甘草酸

18α-甘草酸(18α-GL)是一种齐墩果烷型皂苷,比其差向异构体18β-GL极性小、亲脂性好,更强的抗毒抗炎作用和更高的肝脏靶向性使18α-GL成为保肝护肝领域的主要药物成分。但目前18α-GL的制备方法污染大、效率低,亟需开发一种绿色简单地合成18α-GL的方法。利用酵母细胞异源表达糖基转移酶,通过全细胞催化方式鉴定出可催化18α-甘草次酸(18α-GA)特异性合成18α-单葡萄糖醛酸甘草次酸(18α-GAMG)的糖基转移酶cGuCSyGT和催化18α-GAMG特异性合成18α-GL的糖基转移酶GgUGT1。进一步运用蛋白质结构预测和分子动力学模拟探究cGuCSyGT对18α-GA的催化活性比18β-GA低的原因。最后采用优化底物添加浓度、底盘细胞、底物添加时间、反应时间、培养基成分补加和底物溶剂的策略构建了酵母催化合成18α-GAMG和18α-GL的最优工艺,使18α-GAMG和18α-GL的产量分别达到(36.38±1.87)mg/L和(39.32±0.75)mg/L。本研究实现了18α-GAMG和18α-GL的微生物催化合成,可为18α-GL的微生物全合成提供理论基础和技术支撑。

戴尔有孢圆酵母与酿酒酵母细胞间接触对‘赤霞珠’葡萄酒风味品质的影响

【目的】利用酿酒酵母(Saccharomyces cerevisiae)和非酿酒酵母(Non-Saccharomyces yeasts,NS)混菌发酵可以改善葡萄酒的香气复杂性和浓郁度。深入分析混合发酵剂中酵母细胞之间的接触作用对酒精发酵及代谢产物的调节效应,更好地控制混菌发酵进程。【方法】以预处理的‘赤霞珠’葡萄汁为试材,分别接种戴尔有孢圆酵母(Torulaspora delbrueckii)及S.cerevisiae进行纯种发酵、混菌发酵和双室发酵,分析其发酵动力学及挥发性香气化合物差异;并探究不同接种时间模式下混菌和双室发酵对‘赤霞珠’葡萄酒酿造学参数和风味品质的影响。【结果】T.delbrueckii菌株纯种发酵组的最终还原糖含量为89.00 g·L-1,不能独立完成酒精发酵;S.cerevisiae在纯种发酵、混菌发酵和双室发酵组中均保持较高的生长活力,能顺利完成酒精发酵;但混菌发酵过程中的酵母细胞间接触显著降低了T.delbrueckii菌株的生存能力。与S.cerevisiae纯种发酵相比,混菌发酵组和双室处理组的乙醇含量均有所降低,pH显著下降,总酸显著增加(P<0.05);各处理酒样中检测到挥发酸含量均在0.2—0.7 g·L-1。与S.cerevisiae纯种发酵和双室发酵组相比,混菌发酵组的挥发酸及花色苷含量显著降低。气相色谱-质谱联用技术检测结果显示,T.delbrueckii菌株纯种发酵组香气物质含量最低;相比S.cerevisiae纯种发酵组,混菌发酵和双室发酵组的酯类物质含量均显著增加,高级醇和C6化合物含量明显降低。混菌发酵组中乙酸异戊酯、乙酸己酯、己酸乙酯和辛酸乙酯等酯类化合物含量较双室发酵组显著增加,高级醇和苯衍生物含量明显降低。此外,在混菌发酵过程中,S.cerevisiae的接种时间对乙酸异戊酯和乙酸己酯的生成也有显著影响。感官分析结果表明,与S.cerevisiae纯种发酵相比,同时接种(0 h)混菌发酵处理组显著降低葡萄酒中的生青味,酒样呈现较为浓郁的果香和花香味。【结论】在葡萄酒的混菌酒精发酵过程中,酵母细胞间接触不仅降低T.delbrueckii的生存能力,也显著影响‘赤霞珠’葡萄酒的香气特征和感官品质。

基于改进电阻抗技术的酵母菌细胞活性检测

目的为了使用神经网络快速预测并探究酵母菌细胞活性与电阻抗之间的关系。方法选用电阻抗技术对不同浓度活性酵母菌细胞进行测试,获得死亡细胞的阻抗值。基于灰狼算法优化的BP神经网络的预测模型,探究酵母菌活性与电阻抗在不同频率下的关系。结果酵母菌活性与电阻抗存在复杂的非线性关系,在一定频率下随着酵母菌细胞浓度的增加,酵母菌细胞悬浮液的电阻抗也随之增加。发现活性细胞在相对频率下的电阻抗要高于死亡细胞。基于灰狼算法优化的BP神经网络预测模型误差明显小于BP神经网络,且拟合值更加接近真实值。结论该文所提方法能有效解决电阻抗与酵母菌活性的非线性关系,能够为电阻抗技术在细胞检测领域的应用提供参考。

优化微生物发酵决明子工艺及其保湿功效研究

目的通过微生物发酵技术对决明子进行发酵,从而提高活性成分含量,并探究发酵液在保湿方面的功效。方法根据总蒽醌、总黄酮和总多酚指标筛选酵母菌Y101,采用Plackett-Burman与Box-Behnken实验设计来确定最佳发酵方案,以HaCat细胞干燥损伤模型与AB系斑马鱼脱水模型评估决明子发酵液的皮肤保湿效果。结果以蒽醌含量作为响应值,优化后发酵液中蒽醌含量增加了约2.6倍;决明子发酵液降低了HaCat细胞的干燥死亡率,同时缓解了斑马鱼在高渗透压下的脱水皱缩程度。结论决明子发酵液具有一定的保湿功效,这为其在化妆品和食品领域的应用提供了实验基础和理论支持。

基于酵母双杂交文库鉴定鸡PGCs形成过程中的关键泛素化修饰酶

试验拟筛选与C2EIP互作的蛋白,构建调节PGCs形成过程的泛素化E3连接酶复合体。通过逆转录和同源重组等方法构建鸡PGCs酵母文库;将C2EIP连入诱饵空载体pGBKT7中,并转化酵母感受态细胞进行诱饵载体(C2EIP-pGBKT7)的毒性和自激活检测;将酵母文库与诱饵载体菌液共培养后,通过涂板筛选阳性克隆并进行测序和生物信息学分析筛选互作蛋白。结果显示:成功构建了鸡PGCs酵母文库,其滴度为3.0×10^(6)cfu/mL,总库容为1.5×10^(7)cfu,且重组率为100%,符合筛库要求。成功构建无毒性和不能自激活的C2EIP-pGBKT7诱饵载体,能够用于后续的文库筛选。C2EIP-pGBKT7诱饵载体菌液与酵母文库共培养后,通过PCR测序与蛋白序列分析后共筛选了36个C2EIP的互作蛋白,其中RCJMBM04和CCDC174蛋白功能域分析发现分别具有典型的RING功能域和PPXY基序,进行的生物信息学分析发现WWP1和WWP2具有典型的WW功能域,能够与CCDC174结合。研究表明,试验成功鉴定了C2EIP/RCJMB04/CCDC174/WWP泛素化E3连接酶复合体,为后续研究泛素化修饰调控鸡PGCs形成机制奠定理论基础。