Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.     

Formulation of Protective Agents for Improvement of Lactobacillus salivarius I 24 Survival Rate Subjected to Freeze Drying for Production of Live Cells in Powderized Form

The effectiveness of formulations using different protective agents to maintain viability of Lactobacillus salivarius I 24 during freeze drying for production of live cell in powdered form was investigated. The influence of prefreezing and cultivation conditions on viability of cells after freeze drying was also studied. Surface methodology was used to determine the most suitable combination of the protective agents. Concentrations of skim milk, sucrose, glycerol, and calcium carbonate were selected as operating variables, and survivals of cultures after freeze drying were used as results. Skim milk and sucrose were better protective agents than glycerol and calcium carbonate when used individually for preserving L. salivarius I 24 during freeze drying. Their protective abilities could be enhanced significantly when using them as a mixture (9.85% w/v skim milk and 10.65% w/v sucrose). Prefreezing of the cells at −80°C for 5 h prior to freeze drying and cultivation with regulated pH and temperature gave the highest cell viability.     

Optimization of Dehydration of Lactobacillus salivarius Using Radiant Energy Vacuum

Radiant energy under vacuum (REV) as a rapid dehydration method was employed to dehydrate Lactobacillus salivarius 417 along with different concentrations of skim milk powder as protective medium. Two optimization methods: response surface methodology and random centroid analysis (RCO) were applied and compared to optimize the dehydration parameters for maximum viability—microwave power, absolute pressure in the drying chamber, and protective agent concentration. The study showed that both methods were suitable for the process optimization. Microwave power, concentration of skim milk powder, and absolute pressure had a significant effect on final viability while the speed of sample rotation in vertical axis had no effect (P < 0.05). The result also indicated that a microwave power of <250 W, 10–15% concentration of skim milk powder, and <1 mmHg absolute pressure was needed to achieve viability of equal or greater than 80% in L. salivarius. The RCO-optimized REV process yielded higher viability than the reference freeze drying method and was completed in less than an hour as compared to 64 h for freeze drying.     

Effect of protective agents on the viability of geotrichum candidum during freeze-drying and storage

ABSTRACT:  The effect of 3 cryoprotective agents (trehalose, sucrose, and maltose) on the survival of concentrated cultures of Geotrichum candidum was studied. Initially, the effect of the carbohydrates at 9% and 23% concentrations or combined with skim milk (16%) was compared to the control (skim milk alone) immediately after freeze-drying. Two freeze-drying shelf temperatures, 25 °C and 35 °C, were investigated. Afterwards, the survival of G. candidum freeze-dried with carbohydrates at 23% concentration (alone or combined with skim milk 16%) was studied during 12 wk of storage at 4 °C, in the darkness, under vacuum, and at 0% relative humidity. The glass transition temperature ( T_g ) of the dehydrated protective agents was measured by differential scanning calorimetry. The results showed that the survival after freeze-drying was proportional to the concentration of the protective agents, with skim milk alone giving poor survival of G. candidum . However, when skim milk was combined with disaccharides, a clear improvement was noted. No general tendency of shelf temperature on the survival of G. candidum was noted immediately after freeze-drying. However, changes in the viability were observed during storage. Glass transition temperature ( T_g ) of protective agents linked to their moisture may contribute to predict the stability of lyophilized G. candidum during freeze-drying and storage.     

Comparative evaluation of bacterial cellulose (nata) as a cryoprotectant and carrier support during the freeze drying process of probiotic lactic acid bacteria

Nata or bacterial cellulose produced by Acetobacter xylinum was compared for its cryoprotective and carrier support potential for probiotic lactic acid bacteria against other established cryoprotectants like 10% skim milk, calcium alginate encapsulation or 0.85% physiological saline and distilled water. Individual lactic acid bacteria were grown in MRS broth in the presence of nata cubes or the bacterial suspension mixed with either powdered bacterial cellulose (PBC), 10% skim milk, saline or distilled water and freeze dried. These freeze dried cells were stored at temperatures of either 30 °C or 4 °C and periodically checked for viability. The freeze dried cells on carrier supports were directly used to prepare fermented milks to establish the activity of these cultures. Scanning electron microscopy was employed to visualize the support matrix with the attached lactic acid bacteria. The freeze drying process resulted in a 3.0 log cycle reduction in the colony forming units as compared to the original cell suspension in the case of all the lactic acid bacteria. The growth of lactic acid bacteria in the presence of bacterial cellulose (nata) offers a convenient and easy method to preserve bacteria for short durations and use it as a support to carry out other fermentation processes.     

Optimisation of bambara groundnut water extract and skim milk composition as cryoprotectant for increasing cell viability of Lactobacillus spp. using response surface methodology

Four legume water extracts, that is bambara groundnut, soya bean, red kidney bean and black bean as well as skim milk, were examined for their effectiveness in protecting Lactobacillus rhamnosus GG and Lactobacillus fermentum SK5 during the freeze‐drying and storage. Bambara groundnut water extract (BGWE) showed promising cryoprotective activity that was comparable to skim milk. BGWE and skim milk at 2–10% w/v and 5–20% w/v individually produced survival rates for both strains of 87–88%. To further optimise the synergistic cryoprotective medium, response surface methodology was employed. The optimal combination was 4.93% w/v BGWE and 11.68% w/v skim milk for L. rhamnosus GG and 5.17% w/v BGWE and 11.36% w/v skim milk for L. fermentum SK5 with survival rates of 95.17% and 94.36%, respectively. The storage life of freeze‐dried cells of both probiotics at 4 °C and 30 °C for 6 months was markedly improved when they were produced with these optimal combinations.     

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。     

Fluidized-bed drying as a feasible method for dehydration of Enterococcus faecium M74

The suitability of fluidized-bed technology for the dehydration of probiotic Enterococcus faecium M74 was evaluated. Fluidized-bed drying was processed by layering the microorganisms on spherical pellets. The impact on cell viability of atomizing air pressure, processing temperature and time was investigated. Using 1.5 bar atomizing air pressure, 37 °C processing temperature, and 15 min processing time provided optimal dehydration condition. Changing these values resulted in excessive stress on the cells and affected the cell viability. Next, we compared the impact on cell viability of fluidized-bed drying with that of freeze-drying. Fluidized-bed drying caused more substantial losses of cell viability. However, viability of cells pre-treated with membrane protective agents such as sucrose or skim milk was less affected by fluidized-bed drying than by freeze-drying, resulted in a minor degree of membrane damage after 2 months storage. Comparison of the flow characteristics of freeze-dried cells and fluidized-bed dried cells layered on spherical pellets showed the superior flowability of the latter.     

真空冷冻喷雾干燥下川藏高原冰酒发酵菌剂复合保护剂的配方优化

将冰葡萄渣中分离得到的酿酒酵母,经真空冷冻喷雾干燥制成菌剂,通过单因素和正交实验优化保护剂配方,并对该菌剂进行性能分析。结果表明:复合保护剂最佳配方为甘油5%,蔗糖7%,脱脂乳粉11%,麦芽糊精15%,菌剂产品活菌数和活菌率分别为10.34 CFU/g和78.25%。在此最佳配方条件下,该冰酒发酵菌剂各项性能较优,其颗粒表面光滑平整,具有较强的耐热性和低温稳定性,在10 ℃下能发酵产气,其发酵性能优于市售酿酒酵母。     

喷雾干燥法制备冰酒发酵剂的研究

从四川藏区高原霞多丽冰葡萄渣中分离纯化出了一种菌株,经18S rDNA D1/D2序列鉴定为葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）,并通过正交试验对保护剂的配方进行优化,单因素试验初步研究喷雾干燥的的条件。结果表明,喷雾干燥最佳保护剂的配方为脱脂奶粉7%,酵母浸粉5%,乳糖0.6%,黄原胶0.20%,得到的干菌粉中的活菌数为2.6×1010 CFU/g;确定喷雾干燥实验最适条件为进口温度120 ℃,进样流量20 mL/h,保护剂与菌泥比例4∶1（g∶L）,按上述条件得到的干菌粉的活菌数为2.4×108 CFU/g。     

Foam‐Mat Freeze‐Drying of Bifidobacterium longum RO175: Viability and Refrigerated Storage Stability

Foaming as a pretreatment was used prior to freeze‐drying of Bifidobacterium longum RO175 to investigate the potential acceleration of the drying rate and increase in microorganism viability after the process. A study on storage of foamed and nonfoamed freeze‐dried products at 4 °C completed this study. B. longum RO175 in foamed medium could be freeze‐dried in 1/7 to 1/4 of the time required for nonfoamed suspensions. In addition, foamed suspensions presented higher viability immediately after freeze‐drying (13.6% compared to 12.81 % or 11.46%, depending on the cryoprotective media). Refrigerated storage led to a reduction in B. longum RO175 viability for all tested protective agents (foamed and nonfoamed). No correlation between glass transition temperature and stability of probiotic powders was observed during storage. In addition, lower viability after 56 d of storage was observed for foamed materials, probably due to foam porous structure and higher hygroscopicity, and oxygen presence and moisture pickup during storage.     

猕猴桃酒酵母冻干保护剂配方优化研究

利用JMP和Box-Behnken响应曲面设计(RSM)研究了真空冷冻干燥时脱脂乳粉、海藻糖、蔗糖和抗坏血酸对猕猴桃酒酵母WF_(25)的保护作用。研究结果表明,蔗糖、海藻糖、脱脂乳粉和抗坏血酸均对供试酵母真空冷冻干燥具有保护作用,当它们的添加量分别为121.1、93.9、145.4和10.5 g/L时,活菌率可达到92.21%。     

Effect of diacetyl on the heat stability of concentrated milks

Very low levels of diacetyl (1–5 mM) markedly increased the heat stability of concentrated milks (20% total solids) reconstituted from skim milk powders. Diacetyl caused only slight stabilization of the concentrates (following dilution to 20% total solids) used in the manufacture of these powders but its efficacy was increased by heating the concentrates (70°C for 10 min) prior to diacetyl addition and especially by freeze drying. Laboratory concentrated milks were destabilized by diacetyl and these samples were also destabilized by freeze drying. However, addition of diacetyl prior to preheating and concentration increased stability.     

Drying and storage of crude β-galactosidase extracts from Lactobacillus delbrueckii ssp. bulgaricus 11842

The retention of β-galactosidase activity in crude cellular extract (CCE) preparations from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was investigated after spray drying at three different outlet air temperatures (40, 50 or 60 °C), freeze drying, and after 30 days storage. Lactose, skim milk and whey protein preparations in concentrations ranging from 5 to 30% (w/w) were used as drying adjuncts. To further investigate the protective role of sugars in the enzyme activity preservation, cellobiose and sucrose were also employed in 5 and 10% concentrations during spray-drying at 60 °C or freeze-drying. The addition of lactose or skim milk in all examined concentrations resulted in significantly (P<0.05) higher β-galactosidase activity retention in comparison to all other CCE spray dried at 60°C. The effect was less pronounced at lower spray drying temperatures and increased whey protein concentrations, especially during freeze drying, when almost complete recovery of the enzyme activity upon reconstitution was achieved. Cellobiose provided less β-gal protection in comparison to lactose or sucrose. Lactose was more effective than sucrose at 5% concentration, but both sugars were equal at 10%. The β-gal activity retention in dry CCE preparations during storage at 7 °C over 30 day period was related to the initial water activity; higher initial a_w of powders obtained at lower spray drying temperature was correlated with significant (P<0.05) β-gal activity loss. Freeze dried and spray dried (60 °C) preparations were more stable in comparison to all other samples, retaining high β-gal activity during storage up to 30 days.     

Effect of cryoprotective agents on survival and stability of Lactobacillus acidophilus cultured in food‐grade medium

The effect of cryoprotectants on survival and stability of freeze‐dried Lactobacillus acidophilus ATCC‐4962 cultivated in food‐grade medium was evaluated. The food‐grade medium employed was more economical than the commercial de Man Rogosa and Sharpe medium and gave a higher yield of L. acidophilus ATCC‐4962. Cryoprotective agents improved significantly the cell viability. Skim milk, skim milk with malt extract and monosodium glutamate provided significantly higher viable counts, at optimum concentration of 0.3%. At higher concentration, there was a reduction in cell viability, attributed to cell shrinkage associated with osmotic pressure changes inside the cells. Lactobacillus acidophilus ATCC‐4962 was stable at 28°C until 8 weeks.     

Calcium-Aggregated Milk: a Potential New Option for Improving the Viability of Lactic Acid Bacteria Under Heat Stress

Heat-induced inactivation of viable cells restricts a wide range of application of spray drying in producing dried lactic acid bacteria (LAB) products. In the present study, an effective method to enhance the stability of LAB under heat stress has been identified. This was done by enhancing the heat stability of skim milk as the carrier. Here, skim milk was supplemented with 10 mM CaCl₂ and heated to 90 °C for 10 min to induce protein aggregation. Using this Ca-aggregated skim milk as carrier, the survival of five LAB strains tested was found two orders of magnitudes higher than that of an untreated milk after the heating at a rising temperature from about 25 to 70 °C within 45 s. Possible mechanisms of the protection were explored by comparing the residual viability, microstructure of the cell-contained milk, and changes of suspension particle sizes caused by heat treatment of the four carriers, i.e., untreated milk, Ca-added milk, heat-treated milk, and Ca-aggregated milk. Ca-aggregated milk induced the highest microbial heat stability among them, providing a thick and compact encapsulation around LAB cells before the heat treatment for inactivation. The viable cells could stay in a comparatively more stable extracellular environment. This work reveals potentially a new option for using milk protein aggregates as a protectant of microorganisms. A series of calcium-enriched probiotic products may be developed based on the described principles of the finding of the Ca-aggregated milk.     

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.     

嗜热链球菌M5-5冻干保护剂配方的优化

为了研究不同冻干保护剂在真空冷冻干燥过程中对嗜热链球菌M5-5菌体细胞的保护效果,采用单因素试验和正交试验,以冻干后的菌体存活率为评价指标,考察9种冻干保护剂在冷冻干燥过程中对M5-5菌体的保护效果,以期获得最优保护剂配方。结果表明,海藻糖、甘油和脱脂乳作为单一冻干保护剂时,菌体细胞存活率分别为43.85%、47.51%和40.86%,显著高于其他6种保护剂（P＜0.05）;通过正交试验确定冻干保护剂的最优配方为海藻糖60 g/L、谷氨酸钠20 g/L、甘油20 g/L、脱脂乳150 g/L,此时嗜热链球菌M5-5的冻干存活率可达88.41%。扫描电镜结果显示,添加保护剂配方组菌体细胞完整,表面光滑,说明该保护剂配方能有效减少冷冻干燥过程对菌体细胞的损伤。     

Survival of Bifidobacterium longum 1941 microencapsulated with proteins and sugars after freezing and freeze drying

Five types of proteins and three types of sugars were examined for their effectiveness in protecting B. longum after freeze drying, including their acid and bile tolerance, surface hydrophobicity, retention of β-glucosidase, lactate dehydrogenase and adenosine triphosphatase. Sodium caseinate 12%, whey protein concentrate 12%, sodium caseinate:whey protein concentrate 6%:6%, skim milk 12%, or soy protein isolate 12% was combined with glycerol (3% w/v), mannitol (3% w/v) or maltodextrin (3% w/v). Fifteen emulsion systems containing sugars were obtained. Concentrated B. longum 1941 was incorporated into each emulsion system at a ratio of 1:4 (bacteria:emulsion). All the mixtures were then freeze dried. Water activity (a_w) of freeze dried microcapsules was in the range of 0.30 to 0.35. WPC–CAS GLY provided high stability of bacteria (99.2%) during freezing, while high stability of cells after freeze drying and during exposure to acid and bile environment was achieved when CAS–MAN was applied (97.4%, 81.6% and 99.3%, respectively). High retention of β-glu of freeze-dried bacteria was achieved using SM–MAN as protectant (94.6%). ATPase and LDH were successfully retained by SM–GLY (94.9 and 83.6%, respectively) but there was no significant difference in protection effect using CAS–MAN (93.8 and 82.6%, respectively). Overall, milk proteins were superior to SPI and sugar alcohols provided more protection than MD.     

Lactosylation of milk proteins during the manufacture and storage of skim milk powders

Extensive lactosylation of milk proteins in standard skim milk powder dried against air between 185 and 90°C (inlet and outlet temperatures of the air) was detected by capillary electrophoresis. Optimisation of the drying conditions included keeping the outlet temperature low (preferably <80°C), since this was the parameter which most affected the extent of lactosylation of milk proteins. The inlet temperature was set in order to obtain the best compromise between a low extent of lactosylation and a high drying rate (170–175°C). These conditions allowed the manufacture of low-lactosylated skim milk powder. Storage of freeze-dried and control low-lactosylated skim milk powder at different temperatures showed that both temperature and moisture content affected the progress of lactosylation during storage. Further drying to less than 2.5% moisture content (w/w) and storage at low temperatures were required to prevent the development of lactosylation in the low-lactosylated skim milk powder.