Optimization of the spray drying of a Paenibacillus polymyxa-based biopesticide on pilot plant and production scales

Spore survival and moisture content are two important properties of biopesticides, and both are related to field biocontrol efficacy and storage shelf life. In this study, Paenibacillus polymyxa (HY96-2) was spray-dried on both pilot plant and production scales, and the effects of inlet and outlet temperatures on spore survival and moisture content were investigated. The results showed that inlet temperatures ranging from 170 to 230 °C (at an outlet temperature of 80 °C) had no obvious effect on the two properties during pilot scale processing, although an inlet temperature of 230 °C resulted in higher feed speed. When the outlet temperature on the pilot scale was reduced from 100 to 80 °C, no obvious variations in spore survival and moisture content were found, while a further reduction from 80 to 65 °C resulted in a decline in spore survival from 81.0 to 67.0% and an increase in moisture content from 2.3 to 31.7%. These results indicate that both outlet temperature and moisture content have an effect on spore survival. Optimum inlet and outlet temperatures for P. polymyxa processing were 230 °C and 85–90 °C on a production scale. Under these conditions, spore survival and moisture content were 83.5–86.6% and 2.73––4.12%, respectively.     

点突变对高加索乳杆菌醇脱氢酶酶活的影响

从高加索乳杆菌基因组中克隆醇脱氢酶基因,构建重组表达菌后发现不同转化子具有不同的活性,测序结果表明在部分位点发生了点突变.结合生物信息学知识通过对醇脱氢酶结构与作用机理分析,认为在酶关键位点的变变对酶的活性影响较大,而非关键位点的突变对酶活的影响虽明显降低,但其突变的数目可能对酶活的影响呈现一定的累加效应.其中活性最高的重组菌表达了一个可将苯乙酮高选择对映还原成(S)-笨乙醇的醇脱氢酶,该研究结果为酶的定向进化研究提供了理论依据.     

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto‐aggregation and co‐aggregation capacity. In addition, CS4 and CS7 had greater β‐galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.     

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.     

Preparation and characterization of etoricoxib solid dispersions using lipid carriers by spray drying technique

The basic objectives of this study were to prepare and characterize solid dispersions of poorly water-soluble drug etoricoxib using lipid carriers by spray drying technique. The properties of solid dispersions were studied by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), differential scanning calorimetry (DSC), hotstage microscopy (HSM), radiograph powder diffraction (XRPD), and dissolution studies. The absence of etoricoxib peaks in XRPD profiles of solid dispersions suggests the transformation of crystalline etoricoxib into an amorphous form. In the HSM examination of solid dispersions, the dissolution of drug in the lipid carriers was observed, which was also confirmed by the absence of etoricoxib peak in DSC curves of solid dispersions. The DRIFTS spectra revealed the presence of hydrogen bonding in solid dispersions. The in vitro dissolution rate of solid dispersions as compared with pure etoricoxib, spray-dried etoricoxib, and physical mixtures of drug with lipid carriers. Therefore, the dissolution rate of poorly water-soluble drug etoricoxib can be significantly enhanced by the preparation of solid dispersions using lipid carriers by spray drying technique. Published: October 19, 2005     

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.     

Suitability of buttermilk for fermentation with Lactobacillus helveticus and production of a functional peptide‐enriched powder by spray‐drying

Aim: To ferment buttermilk, a low‐cost by‐product of the manufacture of butter, with a proteolytic strain of Lactobacillus helveticus, to enhance its value by the production of a functional peptide‐enriched powder. Methods and Results: Buttermilk was fermented with Lact. helveticus 209, a strain chosen for its high proteolytic activity. To enhance the release of peptidic fractions, during fermentation pH was kept at 6 by using NaOH, Ca(CO)₃ or Ca(OH)₂. Cell‐free supernatant was recovered by centrifugation, supplemented or not with maltodextrin and spray‐dried. The profile of peptidic fractions released was studied by RP‐HPLC. The lactose, Na and Ca content was also determined. The powder obtained was administered to BALB/c mice for 5 or 7 consecutive days, resulting in the proliferation of IgA‐producing cells in the small intestine mucosa of the animals. Conclusions: Buttermilk is a suitable substrate for the fermentation with Lact. helveticus 209 and the release of peptide fractions able to be spray‐dried and to modulate the gut mucosa in vivo. Significance and Impact of the Study: A powder enriched with peptides released from buttermilk proteins, with potential applications as a functional food additive, was obtained by spray‐drying. A novel use of buttermilk as substrate for lactic fermentation is reported.     

Encapsulation of lemongrass oil with cyclodextrins by spray drying and its controlled release characteristics

Inclusion of the two isomers of citral (E-citral and Z-citral), components of lemongrass oil, was investigated within the confines of various cyclodextrin (α-CD, β-CD and γ-CD) host molecules. Aqueous complex formation constants for E-citral with α-CD, β-CD and γ-CD were determined to be 123, 185, and 204 L/mol, respectively, whereas Z-citral exhibited stronger affinities (157, 206, and 253 L/mol, respectively). The binding trend γ-CD > β-CD > α-CD is a reflection of the more favorable geometrical accommodation of the citral isomers with increasing cavity size. Encapsulation of lemongrass oil within CDs was undertaken through shaking citral:CD (1:1, 1.5:1, and 2:1 molar ratio) mixtures followed by spray drying. Maximum citral retention occurred at a 1:1 molar ratio with β-CD and α-CD demonstrating the highest levels of total E-citral and Z-citral retention, respectively. Furthermore, the β-CD complex demonstrated the slowest release rate of all inclusion complex powders.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Effect of different dextrose equivalents of maltodextrin on oxidation stability in encapsulated fish oil by spray drying

Encapsulating fish oil by spray drying with an adequate wall material was investigated to determine if stable powders containing emulsified fish-oil-droplets can be formed. In particular, the dextrose equivalent (DE) of maltodextrin (MD) affects the powder structure, surface-oil ratio, and oxidative stability of fish oil. The carrier solution was prepared using MD with different DEs (DE = 11, 19, and 25) and sodium caseinate as the wall material and the emulsifier, respectively. The percentage of microcapsules having a vacuole was 73, 39, and 38% for MD with DE = 11, 19, and 25, respectively. Peroxide values (PVs) were measured for the microcapsules incubated at 60 °C. The microcapsules prepared with MD of DE = 25 and 19 had lower PVs than those prepared with MD of DE = 11. The difference in PV can be ascribed to the difference in the surface-oil ratio of the spray-dried microcapsules.     

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (?20 and ?80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at ?80 °C.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.     

静电喷雾法制备乳酸菌微胶囊的实验研究

目的为解决乳酸菌产品活菌数的不稳定性,对乳酸菌进行微胶囊化包埋。方法用海藻酸钠和明胶的混合体系作为壁材,对乳酸菌进行静电喷雾包埋处理,并让微胶囊化乳酸菌在模拟胃肠液的环境中进行耐酸性和肠溶性实验。结果混合体系的壁材与乳酸菌具有较好的生物相容性,优选得出当芯壁材为12时包埋率最高（96．3％）,微胶囊化乳酸菌在经人工胃液处理2h后,活菌数比未经微胶囊化的对照组高出2个数量级,且在经人工肠液处理40min后,乳酸菌几乎全部释放。结论静电喷雾法制备的乳酸菌微胶囊具有一定耐酸性和肠溶性。     

Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir

An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identified as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide. The GC analysis of ZW3 EPS revealed that it was glucogalactan in nature. Exopolymer showed similar flocculation stability like xanthan gum but better than guar gum with a melting point of 93.38 degrees C which is lower than xanthan gum (153.4 degrees C) and guar gum (490.11 degrees C). Compared with other commercially available hydrocolloids like xanthan gum, guar gum and locust gum ZW3 EPS showed much better emulsifying capability.