Intrinsic tolerance of Bifidobacterium species to heat and oxygen and survival following spray drying and storage

AIMS: This study examined the tolerance of various species of the genus Bifidobacterium to heat and oxygen and evaluated the survival of selected strains following spray drying and during storage. METHODS AND RESULTS: Nine Bifidobacterium species were considered to be relatively tolerant to both heat and oxygen and mostly segregated into two clusters within the 16S rDNA phylogenetic tree. Four species were tolerant to oxygen and 12 species were considered sensitive to oxygen and heat. Using a skimmed milk-based carrier good survival following spray drying and storage at 4 degrees C correlated with tolerance to heat and oxygen. Viability was inversely related to storage temperature and at 15 degrees C and 25 degrees C, a significant decline was observed for all species. The inclusion of gum acacia had no significant affect on survival or viability. However, using a fluidized-bed spray dryer viability was greatly improved. CONCLUSIONS: A group of closely related species tolerant to heat and oxygen had high survival following spray drying and maintained viability during prolonged storage at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray drying is a suitable method for the production of skimmed milk powder enriched with high numbers of viable bifidobacteria.     

Preservation of encysted polytomella.

Cysts of Polytomella parva and Polytomella caeca were recovered after 7 days storage at cryogenic temperatures following drying on shredded filter paper, silica gel or without added substrate. Accelerated storage testing, by exposing dried material to elevated temperatures, indicated that shredded filter paper was the best of the substrates tested. Polytomella parva was recovered after 5 years storage at -30 degrees C when dried on filter paper but not when dried on silic agel. Determinations of the number of cysts recovered indicated that viable cysts survived all conditions of storage tested. However, excystment following storage was delayed, the extent depending on storage conditions and the substrate used for drying. Most rapid recovery occurred when cysts were rehydrated immediately after drying, and after storage on filter paper at below -70 degrees C.     

Evaluation of microencapsulation of a Bifidobacterium strain with starch as an approach to prolonging viability during storage

AIMS: To optimize a spray coating process for the production of encapsulated microspheres containing viable Bifidobacterium cells and to determine whether the readily gelatinized modified starch coating used in this study improved bacterial survival in foods or under acid conditions. METHODS AND RESULTS: An air inlet temperature of 100 degrees C was demonstrated to be optimal for the spray drying process, as it afforded good drying, low outlet temperatures (45 degrees C) and resulted in less than 1 log reduction in bifidobacteria numbers during drying. Maximum recovery yields of 30% were obtained after optimizing the air aspiration conditions. The average size of the Bifidobacterium PL1-containing starch microparticles was determined by scanning electron microscopy to be of the order of 5 microm. The starch-coated cells did not display any enhanced viability compared with free PL1 cells when exposed to acid conditions for 6 h or in two dry food preparations over 20 d storage at ambient temperature (19-24 degrees C). Determination of 1491 nucleotides of the 16S rRNA gene from PL1 indicated that it shared 97% homology with a previously sequenced Bifidobacterium ruminantium strain. CONCLUSIONS: Our data demonstrated that, although spray drying is a valuable process for encapsulating bifidobacteria, further work is required to ascertain a more appropriate coating material that will protect this strain against adverse environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of small, uniformly coated microspheres containing viable bifidobacteria using an affordable and industrially convenient process, such as spray drying, has commercial implications for the production of probiotic products. Although popular for use as a coating polymer by the food industry, this study indicated that modified starches might not be suitable for use as an encapsulating material for probiotic strains.     

不同干燥方式、存储温度对羊栖菜中岩藻黄素稳定性的影响

为探究不同干燥方式、不同存储温度和存储时间对羊栖菜中岩藻黄素稳定性的影响,本研究考察了三种不同干燥方式,包括自然阴干、低温烘干和冷冻干燥,对羊栖菜中岩藻黄素稳定性的影响。结果表明采用冷冻干燥所得羊栖菜,含水量低,岩藻黄素含量高,达到699.2μg/g,岩藻黄素保留率为94.7%,远高于自然阴干及低温烘干。因此,冷冻干燥是最佳干燥方法。考察了四个不同温度(-20、4、25、30℃)存储对冻干羊栖菜中岩藻黄素稳定性的影响,结果表明随着存储温度的升高,岩藻黄素的降解率明显增高,因此,-20℃为最佳储存温度。     

Recovery of a marine dinoflagellate following controlled and uncontrolled freezing

A free-living, marine dinoflagellate, Crypthecodinium cohnii, was successfully preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a 1 °C/min cooling rate. After storage for a minimum of 7 days at ?150 °C material frozen by this method demonstrated a 47.7% mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at ?55 °C for 1 hr. This material demonstrated a mean recovery of 30.8% with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after uncontrolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 68% recovery following controlled freezing.The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cause.     

Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR

Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 x 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 x 10(3) CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20 degrees C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% +/- 9% and 23% +/- 4%, respectively, at 4 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.     

Polysaccharide production benefits dry storage survival of the biocontrol agent Pseudomonas fluorescens S11:P:12 effective against several maladies of stored potatoes

Pseudomonas fluorescens S11:P:12 (NRRL B-21133) is a biological control agent able to suppress several potato diseases and sprouting. Notably, it produces a polysaccharide during liquid cultivation, and the objective of this work was to determine the role of this material in the bio-control process. First, the polysaccharide was isolated, purified and identified as marginalan, which accumulated to ~3.3 g/L in cultures. The bioactivity of isolated marginalan applied alone or in combination with washed cells of strain S11:P:12 was tested in potato bioassays of dry rot and pink rot suppressiveness and sprout inhibition. Since the formulation and storage of a dried biocontrol product is preferred for commercial use, the impact of marginalan on cell survival during drying and storage was also studied. Washed bacteria formulated with 0–6.6 g/L polysaccharide were either applied to Hyflo granules, then slowly dried for 24 h with airflow at 50–60% relative humidity, or in 1-µL droplets placed in replicate wells of a micro-plate, then quickly dried for 1 h in a biohazard hood. Both Hyflo and micro-plate dry storage results indicated that marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5–5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected P. fluorescens strains, its benefits to drying and storage survival were again evident (especially after 4°C instead of 25°C storage), but its effects were more subtle than for strain S11:P:12, and dry rot suppression was not impacted.     

The Preservation of Plant Pathogenic Bacteria

Some 160 cultures were preserved by freeze drying, under mineral oil and in soil. After storage for 5 years all freeze dried cultures were viable; most cultures of xanthomonads were viable under oil and in soil; pseudomonads survived well in soil but only moderately well under oil; soft-rotting Erwinia spp. survived poorly but storage under oil was better than in soil; other Erwinia spp. and most Corynebacterium spp. survived well in soil and under oil. The mean half lives in years ( h ) calculated for freeze dried cultures of groups of closely related bacteria were: Erwinia 'chrysanthemi group', 0·40; Erwinia 'carotovora group', 0·51; Pseudomonas 'syringae group', 0·50; Xanthomonas spp., 0·84 years. Estimated half lives for Corynebacterium spp. ranged from 1·8 to 6·5 years. There was no evidence that bacteria which had been in culture for more than 3 years before being freeze dried had a longer storage life than those freeze dried within 3 years of isolation. Cultures of the Pseudomonas 'syringae group'had a longer storage life when freeze dried by Greaves'method ( h = 0·73) than when freeze dried by Annear's method ( h =0·50). There appeared to be no general correlation between half life in storage and either the proportion of cells surviving the freeze drying process or the viable cell count immediately after freeze drying. Most of the variation in the results could be attributed to variation in viable cell count between different ampoules of the same batch of a culture.     

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.
Methods and Results:  Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk ( D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica . The three micro-organisms studied varied considerably in their ability to survive to spray drying processes . Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased.
Conclusions:  Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures
Significance and Impact of Study:  It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.     

Viability of rape (Brassica napus L.) seeds following selection on the basis of newly-emerged radicles then subsequent drying and storage

Germinating rape seeds selected on the basis of newly-emerged radicles (1 ± 0.5 mm) were dried to an equilibrium moisture content (c. 11%) in air at 20°C and 80% relative humidity without loss of viability. Storage life of these low-moisture-content germinating (LMCG) seeds at 15°C was limited to 7 days before viability was significantly reduced. However, viability of LMCG seeds was maintained for 84 days in storage at -20°C. Longer periods in store reduced viability, but 96% of seeds still remained viable after 336 days at - 20°C. Increasing periods of storage at -20°C reduced the subsequent seed longevity at 15°C, indicating a reduction in vigour during storage. Storage under reduced pressure or in a nitrogen atmosphere had little significant effect on seed longevity. Reduction of moisture content below 11% using vacuum drying at a range of temperatures reduced seed vigour.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.     

Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67–85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.     

Granulated preparations of azotobacter in a clay-mineral base

Interaction of Azotobacter chroococcum 20 cells with clay minerals increased their viability at supraoptimal temperatures. Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage. The titers of viable bacteria in the preparations remained 60-70% of the initial level after 12-month storage.     

Long-term preservation of yeast cultures by liquid-drying

Various selected strains from about 20 species of yeasts, which are reported to be sensitive to freeze-drying and liquid-drying, were successfully dried directly from the liquid phase without freezing using a simplified liquid-drying method. All tested cultures proved viable and the majority of the tested strains showed good survival rates after drying. However, different survival levels for different yeasts were observed; generally the sensitivity to drying appeared to be strain-specific. After 1 years' storage at 9°C, no further loss in viability was observed. Accelerated storage testing, for 1 week at 45°, resulted in further loss of viability to various degrees. Yeasts that were filamentous, osmotolerant or psychrophilic appeared to be sensitive to liquid-drying and had relatively lower survival levels than the others. Growth and liquid-drying under microaerobic conditions resulted in improved survival. The dried yeast cultures proved stable and no mutation or loss in desirable characters was detected. The method can be used for the drying and long-term preservation of nearly all yeast genera.K.A. Malik and P. Hoffmann are with the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, W-3300 Braunschweig, Germany     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。     

Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria

Aims:  The present study focuses on the impact of two different drying technologies and the influence of protectants on process survival and storage stability of the two lactic acid bacterial strains Enterococcus faecium and Lactobacillus plantarum .
Methods and Results:  After incubation with the protectants glucose, sucrose, trehalose, and maltodextrin the concentrated bacterial suspensions were subjected to fluidized bed drying and lyophilization and subsequently stored at 4, 22, and 35°C for half a year. Lactobacillus plantarum turned out to be more sensitive to both drying methods than Ent. faecium . Without the addition of a protectant cells of both strains suffered higher losses during fluidized bed drying. Elevated storage temperatures correlate with a higher decline of viable bacterial cells.
Conclusions:  Although survival rates varied between the strains, the nonreducing disaccharides revealed overall best protection for both investigated lactic acid bacteria during processing and storage. The addition of protective carbohydrates can prevent the decline in viability during fluidized bed drying.
Significance and Impact of the Study:  The influence of protectants proved to be species specific and therefore needs to be determined on a case-to-case basis. Survival rates, duration, and energy consumption appear to be the crucial parameters to evaluate the economy of production processes for industrial starter cultures.     

Cold shock during liquid production increases storage shelf-life of Cryptococcus nodaensis OH 182.9 after air-drying

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60-70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C.     

Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR

The effect of refrigerated and frozen storage on the viability of Vibrio vulnificus was evaluated using cell suspensions (1 × 10⁸ CFU/ml). Ethidium bromide monoazide (EMA) was utilized to selectively allow real-time (Rti) PCR amplification of target DNA from viable but not dead cells. Bacterial survivors from the EMA Rti-PCR were evaluated by comparison with the plate count assay following different temperature exposures (− 20 and 4 °C) every 24 h for 72 h. The log CFU values from the EMA Rti-PCR assays were erroneously higher than that from plate counts. DNA amplification was not completely suppressed by EMA treatment of low temperature destroyed cells suggesting that membrane damage was not sufficient to allow effective EMA penetration into the cells. The optimal concentration of sodium deoxycholate (SD) was also determined to enhance discrimination of viable and dead cells following exposure of cells to low temperatures. The use of 0.01% or less of SD did not inhibit the Rti-PCR amplification derived from viable bacterial cells. A rapid decrease of the log CFU was observed with cell suspensions subjected to frozen storage and a slow decline in the log CFU occurred at 4 °C. The combination of SD and EMA treatments applied to cells of V. vulnificus held at − 20 °C and 4 °C resulted in a high level of correlation between the log of CFU (plate counts) and the log of the number of viable cells determined from SD+EMA Rti-PCR.     

Yeasts preservation: alternatives for lyophilisation

The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6?months storage at 4 and 25?°C. None of the yeast cultures showed a significant loss in viable cell count during 6?months of storage at 4?°C upon lyophilisation and preservation in dry rice cakes. During storage at 25?°C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4?months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6?months of storage at 25?°C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4?°C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications.     

Formulation and stabilisation of the biocontrol yeast Pichia anomala

The yeast Pichia anomala has antifungal activities and its potential in biocontrol and biopreservation has previously been demonstrated. To practically use an organism in such applications on a larger scale the microbe has to be formulated and stabilised. In this review we give an overview of our experience of formulating and stabilising P. anomala strain J121 in a wider perspective. The stabilisation techniques we have evaluated were liquid formulations, fluidised bed drying, lyophilisation (freeze-drying) and vacuum drying. With all methods tested it was possible to obtain yeast cells with shelf lives of at least a few months and in all cases the biocontrol activity was retained. Fluidised bed drying was dependent on the addition of cottonseed flour as a carrier during the drying process. In liquid formulations a sugar, preferentially trehalose, was a required additive. These two kinds of microbial stabilisation are easily performed and relatively inexpensive but in order to keep the cells viable the biomaterial has to be stored at cool temperatures. However, there is room for optimization, such as improving the growth conditions, or include preconditioning steps to enable the cells to produce more compatible solutes necessary to survive formulation, desiccation and storage. In contrast, lyophilisation and vacuum drying require a lot of energy and are thus expensive. On the other hand, the dried cells were mostly intact after one year of storage at 30°C. Inevitably, the choice of formulation and stabilisation techniques will be dependent also on the intended use.