Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.     

Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions

The biocontrol potential of the yeast Candida sake (CPA-1) against Penicillium expansum decay of apples under several controlled atmosphere conditions was investigated. In a laboratory trial under different commercial cold storage conditions, increasing concentrations of C. sake improved decay control. A maximum reduction of decay was achieved at 3% O2-3% CO2 atmosphere. It amounted to a 97% lesion reduction after treatment with a suspension containing 2.4 x 10(6) CFU/ml of C. sake (CPA-1). In a semi-commercial trial at 1 degree C with wounded fruits, the reduction in decay diameter caused by C. sake exceeded 80% after 60 days at 21% O2 and 60% after 120 days of storage under controlled atmosphere conditions. For seven controlled atmosphere conditions studied, a significant influence by C. sake on the P. expansum decay was observed, and the lesion size was reduced more than 70% by C. sake at 10(7) CFU/ml. The populations of C. sake (CPA-1) on the apple surface followed the same pattern under all controlled atmosphere conditions studied. They decreased 4-10-fold during the first 2 weeks, followed by an increase to the initial level after 45 days, and thereafter the count remained constant for the period of 90 days examined. This indicated the capacity of C. sake (CPA-1) to colonize the surface of apples under various storage conditions. The ability to colonize was even higher in apple wounds.     

Effect of biocontrol agents Candida sake and Pantoea agglomerans on Penicillium expansum growth and patulin accumulation in apples

Penicillium expansum is the major responsible of fruit pome decaying in cold storage. Apples spoiled by P. expansum are expected to contain patulin, a mycotoxin which is proven to affect human health. The use of chemicals is the most common procedure to prevent rots in postharvest but legislation is becoming more and more restrictive. The use of biocontrol agents (BCA) as an alternative tool is currently being proposed. The aim of this study was to evaluate the effect of two BCA (Candida sake CPA-2 and Pantoea agglomerans CPA-1) on P. expansum growth and patulin accumulation in cold storage and further deck (ambient) storage. Wounded apples were inoculated with a cell suspension of either C. sake or P. agglomerans and with a P. expansum conidial suspension. Apples were cold stored at 1 degrees C until lesion diameter reached 2 or 4 cm. Half the apples of each treatment were further stored at 20 degrees C for three days before patulin analyses. Both BCA tested controlled blue rot and patulin accumulation during cold storage. The control of P. expansum growth was enhanced in C. sake treated apples. On the other side, control of patulin accumulation in P. agglomerans treated apples seemed to be more efficient. BCA treatment could not control blue rot and patulin accumulation during further storage at room temperature and in some cases, an increase in P. expansum aggressiveness was observed.     

Control of Penicillium expansum and Botrytis cinerea on apples and pears with the combination of Candida sake and Pantoea agglomerans.

The effectiveness of Candida sake (CPA-1) in combination with Pantoea agglomerans (CPA-2) for controlling Penicillium expansum and Botrytis cinerea on pears and apples was determined. The concentrations tested were 2 x 10(6) and 2 x 10(7) CFU/ml for C. sake and 2 x 10(7) and 8 x 10(7) CFU/ml for P. agglomerans. At room temperature, the two antagonists were combined in proportions of 0 to 100% in 25% increments. At the proportion of 50:50, no rot development was observed in pears, and the greatest control of blue mold in apples was observed at this proportion for all the tested concentrations. Under cold temperature on pears, the highest effectiveness of the mixture was observed when C. sake at 2 x 10(7) CFU/ml was combined with P. agglomerans at 2 x 10(7) or at 8 x 10(7) CFU/ml at the proportion 50:50. Under these conditions, no rot development of blue mold was reported, and gray mold lesion size was reduced by more than 95%. On apples, the mixture of C. sake at 2 x 10(7) CFU/ml and P. agglomerans at 8 x 10(7) CFU/ml at the proportion 50:50 reduced blue and gray mold incidence by 90%. Populations of the two antagonists had the same growth pattern at 20 degrees C when they were applied individually or in combination, but the population level was always higher when they grew alone. In contrast, at 1 degrees C, the population of both antagonists in combination formed a stable community with the same levels as individual application during the first 30 days; after that, C. sake dominated, and P. agglomerans decreased on apples and pears. At both temperatures, the maximum population level of C. sake was observed in apples, and themaximum population level of P. agglomerans was observed in pears.     

真空冷冻干燥法制备益生菌粉的冻干保护剂配方优化

为提高益生菌冻干粉存活率。本文采用真空冷冻干燥法,分别以植物乳杆菌SC1、凝结芽孢杆菌XP2、酿酒酵母菌SA1为实验菌株,研究冻干保护剂脱脂乳粉、低聚木糖、可溶性淀粉和V_C钠盐对三种益生菌冻干存活率的影响。通过单因素和正交试验筛选出最优组合。结果表明经-70℃预冷冻2 h,-50℃,10 Pa条件下冷冻干燥38 h,保护剂最佳配方组合分别为,植物乳杆菌:可溶性淀粉10%,V_C钠盐3%,脱脂乳粉12%,低聚木糖14%;凝结芽孢杆菌:低聚木糖8%,V_C钠盐4%,可溶性淀粉14%,脱脂乳粉8%;酿酒酵母菌:低聚木糖10%,V_C钠盐2%,脱脂乳粉12%,可溶性淀粉12%。添加最优保护剂组合,三株菌的冻干存活率分别为83.2%、83.7%和86.7%,活菌数均高于1.0×108 CFU/g,具有较好的应用价值。     

Survival of Pantoea agglomerans strain CPA-2 in a spray-drying process.

Spray drying could be a suitable method for preserving microorganisms, as it allows large quantities of cultures to be dried at low cost. The aims of this paper were to evaluate the effects of spray-drying conditions on survival of the biocontrol agent Pantoea agglomerans CPA-2, which has shown antifungal activity against Penicillium expansum and Penicillium digitatum on citrus fruits. Various compounds cited in the bibliography as carriers were tested in our spray drying, and some salts (MgSO4, K2SO4. and Na2CO3) and dairy products (lactoserum or nonfat skimmed milk [NFSM]) showed the best results in terms of recovered powder. Outlet temperature had more influence on the death of bacteria than inlet temperature. P. agglomerans was heat sensitive, and the activation energy was around 6 kcal/mol K when MgSO4 (10%) or NFSM (10%) were used as carriers and only 3 kcal/mol K when the combination of MgSO4 (10%) and NFSM (10%) was used. The highest powder recovery was obtained when NFSM was used as the rehydration medium. Although the percentage of powder recovery was not high (around 50%) and viability was low, the results suggest that with bigger spray dryers, we could expect a lower outlet temperature and probably an increased viability. Further research into spray-dryer design is needed in order to demonstrate this.     

不同干燥方式对金针菇品质及多酚氧化酶活性的影响

本研究以新鲜金针菇子实体为材料,采用恒温热风干燥、变温热风干燥、真空冷冻干燥、自然干燥和微波干燥对其进行处理,研究不同干燥方法对干制品感官品质、复水率、膨化率、粉碎率,营养成分以及多酚氧化酶活性的影响。结果表明:真空冷冻干燥效果最佳,自然干燥次之。其中真空冷冻干燥的金针菇干制品的复水率为358.75%,粉碎率为76.37%,膨化率为84.52%,粗蛋白12.07 mg/g,粗多糖34.22 mg/g,维生素C含量为17.18 mg/100 g。自然干燥产品的多酚氧化酶活性最高,酶活性为32.9 U/min,高于其他干燥方法。综上,真空冷冻干燥对金针菇品质影响最低,多酚氧化酶活性最低,适宜用来对金针菇进行干燥处理。     

CaCl2增强橄榄假丝酵母对苹果果实采后青霉病的生防效力

采用同孔接种的方法研究橄榄假丝酵母（Candida oleophila）结合CaCl2处理对采后苹果青霉病的控制效果及相关机理。结果表明：采用1×107 CFU/mL橄榄假丝酵母结合10 g/L CaCl2处理苹果果实,对其采后青霉病有显著的控制效果,且效果明显优于橄榄假丝酵母单独处理;在25 ℃条件下培养,橄榄假丝酵母能在果实伤口处迅速定植生长,并持续保持较高水平,10 g/L CaCl2对橄榄假丝酵母生长状态有明显促进作用;同时,橄榄假丝酵母复合CaCl2处理能显著诱导苹果果实几丁质酶和β-1,3-葡聚糖酶活力的提高,促进相关代谢产物木质素、总酚、类黄酮的积累。     

Integration of biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum

Forty-nine compounds currently used as additives in foods were tested in combination with three biocontrol agents, the yeasts Rhodotorula glutinis, Cryptococcus laurentii, and the yeastlike fungus Aureobasidium pullulans, to increase their antagonistic activity against Penicillium expansum, the causal agent of blue mold on apples. Twelve additives dramatically improved the antagonistic activity of one or more of the tested biocontrol agents. In a two-way factorial experiment with these selected additives the percentage of P. expansum rots on apples was significantly influenced by the antagonist and the additive as well as by their interaction. The combination of the biocontrol agents and some additives resulted in a significantly higher activity with respect to the single treatments applied separately, producing additive or synergistic effects. Some of the selected additives combined with a low yeast concentration (106 cells per ml) had comparable or higher efficacy than the biocontrol agents applied alone at a 100-fold higher concentration (10(8) cells per ml). Some organic and inorganic calcium salts, natural gums, and some antioxidants displayed the best results. In general, the effect of each additive was specific to the biocontrol isolate used in the experiments. Possible mechanisms involved in the activity of these beneficial additives and their potential application in effective formulations of postharvest biofungicides are discussed.     

Tracking Amazonian cheese microbial diversity: Development of an original,sustainable, and robust starter by freeze drying/spray drying

Marajó cheese made with raw buffalo milk in the Amazon region of Brazil can be considered a good source of wild lactic acid bacteria strains with unexplored and promising characteristics. The aim of this study was to develop a potential probiotic starter culture for industrial applications using freeze drying and spray drying. A decrease in the survival rates of freeze-dried samples compared with spray-dried samples was noted. The spray-dried cultures remained approximately 10⁹ cfu·g⁻¹, whereas the freeze-dried samples showed 10⁷ cfu·g⁻¹ after 60 d of storage at 4°C. All of the spray-dried samples showed a greater ability to decrease the pH in 10% skim milk over 24 h compared with the freeze-dried samples. The spray-dried samples showed a greater resistance to acidic conditions and to the presence of bile salts. In addition, under heat stress conditions, reduction was under 2 log cycles in all samples. Although the survival rate was similar among the evaluated samples after drying, the technological performance for skim milk showed some differences. This study may direct further investigations into how to preserve lactic acid bacteria probiotics to produce spray-dried starters that have a high number of viable cells that can then be used for industrial applications in a cost-effective way.     

嗜酸乳杆菌冻干影响因子的研究

传统的乳酸菌发酵剂因活化过程复杂,极易引起污染而影响发酵乳的质量。目前,多采用冷冻干燥技术制备直投式粉末状乳酸发酵剂。然而,菌种冻干时机的把握和冻干保护剂的选择,将直接影响乳酸菌在冷冻干燥前后的存活率以及存活期。本文研究了嗜酸乳杆菌的生长曲线,选用蔗糖、乳糖、甘油、VC、谷氨酸钠、麦芽糊精、脱脂乳粉等为嗜酸乳杆菌冻干保护剂进行了冻干研究。结果表明:①嗜酸乳杆菌发酵液培养至酸度为100°T、pH值4.8时,OD410nm=0.511为最大,是最佳发酵终止时间;②嗜酸乳杆菌冻干保护剂配方以5%麦芽糊精、2%VC、5%甘油为最佳冻干保护剂组合,此时嗜酸乳杆菌冻干菌粉活菌数为2.34×109cfu/g。     

牡蛎冷冻干燥后复水特性及微观结构的研究

研究牡蛎冷冻干燥后的复水特性,将新鲜牡蛎分为直接冻干组(Fresh+FD组)、预煮处理后冻干组(Boiled+FD组)和自然干燥组(Fresh+ND组),考察干燥后牡蛎的干燥比、复水比、复水率和微观结构的差异。结果表明:两冻干组牡蛎的含水量远低于自然干燥组,在常温和沸水中的复水比和复水速率均大于自然干燥组,冷冻干燥更有利于牡蛎中水分的去除和复水;干燥后牡蛎闭壳肌微观形态优劣程度依次为Fresh+FD组>Boiled+FD组>Fresh+ND组,其中Fresh+FD组闭壳肌纤维细长、舒展,间隙均匀,Fresh+ND组闭壳肌纤维束堆叠,组织致密;常温复水80min,两冻干组牡蛎的复重系数和复水率均高于自然干燥组。     

Effect of pulsed vacuum and ultrasound osmopretreatments on glass transition temperature, texture, microstructure and calcium penetration of dried apples (Fuji)

The effect of pulsed vacuum and ultrasound pretreatments on glass transition, texture, rehydration, microstructure and other selected properties of air- and freeze-dried apples (Fuji) were investigated. Apple cylinders (15 mm height × 15 mm diameter) were first osmoconcentrated in a 60 g/100 g high-fructose corn syrup solution containing 7.5 g/100 g Gluconal Cal^® combined with agitation, pulsed vacuum (PV), or ultrasound for 3 h, then hot-air or freeze dried. Changes in glass transition temperature, hardness, crispness, and rehydration rate were measured, microstructure was observed using scanning electronic microscopy (SEM), and calcium ions distributions were analyzed by a laser ablation inductively coupled mass spectrometry (LA-ICP-MS). Under the same drying method, ultrasound led to a higher glass transition temperature, lower water activity, moisture content and rehydration rate, severer structure collapse, less cavities and calcium uptake than PV did. Different osmoconcentration pretreatment had no significant (P < 0.05) effect on the hardness, crispness, shrinkage and rehydration rate. Compared to hot-air drying, freeze-dried apples showed the porous structure, minimal shrinkage, softer texture, better rehydration capacity, lighter color, and slightly lower glass transition temperature.     

枯草芽孢杆菌Prob1822复合抗热保护剂的研究

以枯草芽孢杆菌（Bacillus subtilis）Prob1822为研究对象,以热诱激处理（55 ℃热处理10 min）稳定期菌体存活率为评价指标,通过单因素试验和响应面法研究枯草芽孢杆菌Prob1822复合抗热保护剂的组成。结果表明,单一抗热保护剂以海藻糖、蔗糖及脱脂奶粉抗热保护效果较好;利用响应面法优化复合抗热保护剂配方为海藻糖9.0%、蔗糖5.0%和脱脂奶粉6.8%。在此最优条件下,菌体存活率为（95.24±0.84）%。糖类与蛋白质联用作为复合抗热保护剂比单一保护剂抗热保护效果更好,可减轻喷雾干燥对菌体亚细胞结构及生物大分子损伤,实现枯草芽孢杆菌的抗热保护。     

Efficacy of Candida sake CPA-1 formulation for controlling Penicillium expansum decay on pome fruit from different Mediterranean regions

The effectiveness of a formulated product of the yeast Candida sake CPA-1 for controlling postharvest diseases on pome fruits was demonstrated in laboratory, semicommercial, and commercial trials carried out in the major pome fruit producing region of the European Union. First, one wettable powder and seven liquid formulations were tested in laboratory trials that involved two varieties of apples and two varieties of pears. In all cases, an efficacy similar to that of fresh cells was demonstrated in the control of artificial Penicillium expansum infection. After these trials, the formulated product chosen for semicommercial and commercial trials was LF1, a liquid formulation that is particularly suitable for commercial applications. In semicommercial trials, LF1 showed a performance similar to fresh cells in most trials, and the population dynamics of both fresh and formulated cells were quite stable throughout the storage period. This indicates the high viability of C. sake CPA-1 in this formulation and the absence of adverse effects during the formulation of the product, which may significantly affect both its ability to grow on fruit and its antagonistic activity. We evaluated the control of natural infection after applying the formulated product in a commercial drencher in different packinghouses. A significant reduction in the incidence of diseases was observed with a recommended dose of around 10(7) CFU/ml when natural infections were greater than 1%. In general, large quantities of yeast were observed on the surface of unwounded fruits of different pome fruit cultivars. Moreover, populations of this biocontrol agent increased rapidly on fruit surfaces and remained quite stable for a long time under commercial storage conditions. Commercial practices used in packinghouses were therefore successfully applied for this formulated product.     

Effects of enzymatically-extracted purple rice bran fiber as a protectant of L. plantarum NRRL B-4496 during freezing,freeze drying,and storage

This study investigated purple rice bran fiber (PRF) as a protectant for Lactobacillus plantarum NRRL B-4496 (LP) during freezing, freeze drying and storage. PRF was enzymatically extracted from purple rice bran. L. plantarum NRRL B-4496 was grown in MRS broth, centrifuged, and immobilized on PRF suspension. LP cells immobilized on PRF (LP-PRF) and free LP cell (control) samples were frozen in either air blast (AF) or cryogenic freezing (CF) before freeze drying. Freeze-dried (FLP) samples were stored either at room temperature or at refrigerated temperature. For either freezing method, PRF protected cells had less than one log reduction of viable cells while the control had reductions greater than six logs after freeze drying. The log reductions of viable LP cells protected with PRF after freeze drying and 12 weeks storage at 4° C for AF and CF treatments were 0.7 and 1.3 log cycle, respectively. The viable LP-PRF cell count for CF was significantly lower than for AF after 12 weeks at room temperature. PRF improved LP survival in both AF and CF samples in bile. This study demonstrated that freezing methods affected LP viability during storage and that PRF could protect at both refrigerated and room temperatures.     

Chemical composition and nutritive value of spray dried permeate powder

Reconstituted skimmed milk (10% TS) was ultrafiltered, and the permeate was concentrated threefold by reverse osmosis and then spray dried at different temperatures (145–195° C). The drying conditions had no effect on the moisture content or the major constituents of permeate powder. The powder was mainly lactose (85%) with 6.35460% ash. A small percentage of the lactose in the permeate powder was present as a-lactose. Sodium, potassium and citrate were present in high concentrations nearly equivalent to their average percentage in skimmed milk. The calcium, magnesium and inorganic phosphorus contents of permeate powder were also determined. The thiamine, riboflavin and niacin contents of permeate powder averaged 0.445, 0.947 and 0.729 mg/100 g respectively. The drying temperatures had no effect on the vitamin contents of the powder. The permeate powder had average Fe, Zn, Mn and Cu contents of 94.7, 21.1, 3.1 and 9.5 Fg/100 g respectively.     

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.     

The Viability of Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM Following Double Encapsulation in Alginate and Maltodextrin

Lactobacillus rhamnosus GG (LGG) and Lactobacillus acidophilus NCFM (LNCFM) were encapsulated in alginate microgel particles (microbeads) by a novel dual aerosols method. The encapsulated probiotics in microbead gel matrix were further stabilized in maltodextrin solids by either spray or freeze-drying to form probiotic microcapsule powders. The free cells of probiotics were also sprayed and freeze-dried in maltodextrin only without microgel encapsulation. After rehydration of microgel-encapsulated powder, gel particles regained their shape. There was no difference in the loss of viability between encapsulated and unencapsulated probiotics during spray drying or freeze-drying. For LNCFM, spray-dried bacteria with or without gel encapsulation exhibited less death (3.03 and 3.07 log CFU/g reduction, respectively) than those of freeze-dried bacteria (4.36 and 4.89 log CFU/g reduction, respectively) after 6 months storage at 4 °C. The same trend was also observed in spray-dried LGG without gel encapsulation which showed 5.87 log CFU/g reduction in viability after 6 months at 4 °C; however, freeze-dried LGG without gel encapsulation exhibited a rapid reduction in viability of 5.91 log CFU/g within just 2 months. Gel-encapsulated LGG which was freeze-dried exhibited less death (3.32 log CFU/g reduction) after 6 months at 4 °C. This work shows that spray drying results in improved subsequent probiotic survivability compared to freeze-drying and that alginate gel encapsulation can improve the survivability following freeze-drying in a probiotic-dependent manner.     

高活性益生菌发酵枸杞粉的真空冷冻干燥工艺优化

以植物乳杆菌发酵枸杞浆为研究对象,通过差示扫描量热仪和冻干显微镜分析益生菌发酵枸杞浆的物性参数,采用正交试验对保护剂进行选择和优化,并绘制冻干曲线,旨在开发出一套制备富含活性益生菌的发酵枸杞粉的工艺方法。结果表明：运用差示扫描量热仪,采用经过退火处理的连续扫描法,在退火温度－5 ℃、升温速率5 ℃/min条件下,测得的益生菌发酵枸杞浆物料的物性参数为：共融点－5.28 ℃,结晶点－19.59 ℃,玻璃态转化温度－31.82 ℃,崩解温度约－36 ℃。根据已确立的参数确定发酵枸杞浆的预冻温度为－40 ℃,预冻时间为4 h,升华干燥阶段的温度为－45 ℃。最佳复配保护剂组合为：20%麦芽糊精、14%乳糖、6%海藻糖。复配后样品的升华干燥温度为－30 ℃,分别绘制真空冷冻干燥过程中的冻干曲线。益生菌发酵枸杞冻干粉后活菌数达9.6（lg（CFU/g））,含水量低于3%,色泽完好,复水性强,产品质量佳。