Spray-dried Cells Research Articles

Whey protein hydrolysates as prebiotic and protective agent regulate growth and survival of Lactobacillus rhamnosus CICC22152 during spray/freeze-drying, storage and gastrointestinal digestion.

Probiotic products are receiving increasing attention because of their tremendous beneficial health effects. However, it is still a great challenge to preserve probiotic viability during processing, storage and gastrointestinal digestion. Encapsulation is a widely known technology for enhancing bacterial viability and product stability. Hence highly hydrolyzed whey protein hydrolysate (HWPH) and moderately hydrolyzed whey protein hydrolysate (MWPH) used as a one-step culture medium and wall material for Lactobacillus rhamnosus were investigated. H/MWPH-substitutive medium for the growth of Lactobacillus rhamnosus presented double the biomass production compared to other media. The H/MWPH-substitutive medium in combination with freeze drying also led to the highest survival ratio (97.13 ± 9.16%) and cell viability (10.62 log CFU g-1 ). The highest survival rate of spray-dried cells was 85.56 ± 7.4%. In addition, the cell viability of spray-dried Lactobacillus rhamnosus with MWPH as culture and dry medium was 0.79 log CFU g-1 higher than that of HWPH. Images confirmed that spray-dried Lactobacillus rhamnosus in MWPH provided better protection and it showed greater sustained viability after gastrointestinal digestion. Overall, WPH just as carrier provides better thermal protection and MWPH is a preferable two-in-one medium for probiotics. © 2022 Society of Chemical Industry.

Multi-layer PLGA-pullulan-PLGA electrospun nanofibers for probiotic delivery

Encapsulation of bacteria into a polymer matrix can potentially enhance the delivery of probiotics. In this study, we present a multilayer electrospun construct to facilitate enhanced delivery of probiotics, where the internal layer is loaded with bacterial cells and the external layers sandwich the internal layer for increased protection and potential mucoadhesion properties. In this proof of concept study, Lactobacillus rhamnosus GG (LGG), which is a robust and well-studied probiotic strain, was encapsulated into pullulan nanofibers, with two electrospun PLGA (Poly-lactic-co-glycolic acid) layers covering it. According to our in vitro study, there was a large decrease in the viability of LGG released from the monolayer sample (LGG:pullulan). However, the multilayer construct maintained excellent viability and an acceptable storage potential. Our in vivo competition study showed that LGG, delivered by multilayer construct, were able to survive intestinal transit and were recovered from all segments of the intestine. Not only did the multilayer construct perform as well as non-encapsulated spray-dried cells in terms of viability and establishment of LGG, but it also showed some cases of increased LGG colonization from fibers in jejunum and cecum compared to spray-dried LGG three days after dosage. We believe, the multilayer construct has the potential to enhance the delivery of the strains that require additional protection, and can benefit from mucus embedment with the help of the covering layers. Particularly, the use of electrospun fibers for the protection of next-generation probiotics sensitive to oxygen and/or gastric conditions could have major commercial interest.

Method of preservation and type of protective agent strongly influence probiotic properties of Lactococcus lactis: A complete process of probiotic preparation manufacture and use

In order to assess the essential probiotic properties of a strain dedicated for administration in humans and animals, characteristics of finally formulated products, rather than the cells solely, seems to be of crucial importance. In this study, composition of protective blends for manufacture of L. lactis probiotic powders was optimized using a statistical experimental design. The powders, generated by either spray- or freeze-drying techniques, were subsequently subjected to storage testing, and in vitro digestion in simulated stomach and small intestine. Finally, maintenance of adherence capability to human enterocyte-like cell lines, was evaluated. Our data demonstrated that 10% trehalose ensures the highest viability of L. lactis bacteria upon both drying techniques (viability of 60–68%). Moreover, skimmed milk-protected spray-dried cells exhibit the highest resistance to harsh environmental conditions of stomach (53.9 ± 7.6% survival rate) and higher adhesion ability to HT-29 cell line after digestion (528 ± 29 cells per 100 epithelial cells).

Survival of spray-dried and free-cells of potential probiotic Lactobacillus plantarum 564 in soft goat cheese.

A high viability of probiotics in food product, with a living cells threshold of 107 /cfu/g (colony-forming units/g) is a challenge to achieve in food production. Spray drying is an efficient and economic industrial method for probiotic bacterial preservation and its application in food products. In this study, the survival of free and spray-dried cells of potential probiotic strain Lactobacillus plantarum 564 after production and during 8weeks of storage of soft acid coagulated goat cheese was investigated, as well as compositional and sensory quality of cheese. Total bacterial count of spray-dried Lb. plantarum 564 cells were maintained at the high level of 8.82log/cfu/g in cheese after 8weeks of storage, while free-cell number decreased to 6.9log/cfu/g. However, the chemical composition, pH values and sensory evaluation between control cheese (C1 sample made with commercial starter culture) and treated cheese samples (C2 and C3, made with the same starter, with the addition of free and spray-dried Lb. plantarum 564 cells, respectively) did not significantly differ. High viability of potential probiotic bacteria and acceptable sensory properties indicate that spray-dried Lb. plantarum 564 strain could be successfully used in the production of soft acid coagulated goat cheeses.

Effects of spray drying on Lactobacillus plantarum BM-1 viability, resistance to simulated gastrointestinal digestion, and storage stability

ABSTRACTThe aim of this study was to evaluate the applicability of spray drying in the production of high-viability powder of L. plantarum BM-1. Firstly, in order to improve the survival of L. plantarum BM-1 during spray drying, different protectants were added before drying. The results showed that the highest survival rate of 75.70% and the lowest moisture content of 3.67% were achieved with the combination of reconstituted skim milk (RSM) and equal weight sucrose as protective agent. The cell counts reduction of free L. plantarum BM-1 was 2.58 log CFU/ml after exposure to 60°C for 1 min, whereas the reduction in spray-dried cells protected by RSM and sucrose was only 0.08 log CFU/ml. After 120-min incubation in simulated gastric condition, cell counts of free L. plantarum BM-1 decreased approximately 1.4 log CFU/ml, whereas no significant (p > 0.05) reduction in spray-dried cells was observed. Compared to the free cells, the spray-dried cells also showed higher survival under bile salts stress. In addition, the bacteriocin production of L. plantarum BM-1 was not affected during spray drying. Furthermore, the survival rate of spray-dried powder reached 98% after storage for 2 months at 4°C, and a nitrogen replacement package was shown to be better than a vacuum package and air-sealed package at room temperature. Therefore, the combination of equal weight RSM and sucrose is a promising protective agent for L. plantarum BM-1 during spray drying.

The Ability of Lactobacillus rhamnosus in Solution, Spray-Dried or Lyophilized to Bind Aflatoxin B1

<p>Aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) can cause carcinogenic, mutagenic, teratogenic and immunosuppressive effects in humans and animals. Several lactic acid bacteria species have the ability to bind AFB<sub>1 </sub><em>in vitro</em>, showing a potential application for reducing the bioavailability of AFB<sub>1</sub> in contaminated products. Thus, the aim of this study was to evaluate the capacity of <em>Lactobacillus rhamnosus</em>, non-viable and dried, in removing the AFB<sub>1</sub> from a contaminated medium. <em>L. rhamnosus</em> were cultured in MRS broth, sterilized (121 ºC, 15 min.) to inactivate their metabolism and then dried by spray-drying or freeze-drying (lyophilization). Binding assays using AFB<sub>1</sub> (1.0 µg/ml) and <em>L. rhamnosus</em> cells (1×10<sup>10</sup> cells, in suspension or spray-dried or freeze-dried) were conducted at pH 3.0 and 6.0, room temperature and contact time of 60 min. Quantification of AFB<sub>1</sub> was achieved by high performance liquid chromatography. Scanning electron microscope was also performed in order to analyze the drying effect on the atomized and lyophilized <em>L. rhamnosus</em> cells. For pH 3.0 and 6.0, there were no significant differences between AFB<sub>1</sub> binding efficiency by <em>L. rhamnosus</em> cells in solution (45.9 ± 8.8% and 35.8 ± 7.7%, respectively) or freeze-dried (36.6 ± 7.1% and 27.2 ± 4.0%, respectively). However, the spray-dried cells lost completely the AFB<sub>1</sub> binding capacity during atomization, which damaged the structural and functional properties of the bacterial cell wall. In conclusion, <em>L. rhamnosus</em> retained its AFB<sub>1</sub> binding ability only when its cell wall remained intact as observed in the lyophilization procedure. Lyophilized <em>L. rhamnosus</em> cells therefore can be a practicable alternative for decontamination of food products susceptible to aflatoxin contamination.</p>

Non-viable Lactobacillus reuteri DSMZ 17648 (Pylopass™) as a new approach to Helicobacter pylori control in humans.

Prevalence of infections by Helicobacter pylori, a pathogen involved in a number of gastrointestinal diseases, remains high in developing countries. Management of infections by eradication is not always an option. Lactobacillus reuteri (L. reuteri) DSMZ17648 (Pylopass™/Lonza) specifically co-aggregates H. pylori in vitro and was shown to reduce 13C urea breath test in vivo. In this pilot study, we tried to replicate previous findings in an independent sample and to evaluate effects of spray-drying vs. freeze-drying of cultures. A single-blinded, placebo-controlled study was done in 22 H. pylori positive, asymptomatic adults. H. pylori levels were determined by 13C-urea-breath method after 14 days of supplementation, as well as after 6, 12, and 24 weeks follow-up. In the test group, but not in the placebo group, a significant reduction of H. pylori was observed. For the first time, spray-dried cells of L. reuteri DSMZ17648 have been used in a human study and results are in line with the first study results, supplementing with freeze-dried material. This is of special interest as spray-drying results in dead cell material, meaning that the effect of L. reuteri must be independent of its probiotic activity. These results confirm the potential of Pylopass™ as a novel way to reduce the load of H. pylori.

Characterization of free and immobilized (S)-aminotransferase for acetophenone production

Enzyme immobilization often improves process economics, but changes in kinetic properties may also occur. The immobilization of a recombinant thermostable (S)-aminotransferase was made by entrapment on calcium alginate-3% (w/v)-and tested with (S)-(-)-(alpha)-methylbenzylamine for acetophenone production. The best immobilization results were obtained for beads of concentration of 10 mg of spray-dried cells (containing recombinant (S)-aminotransferase) per milliliter of sodium alginate bead. As a result of immobilization, the properties of immobilized spray-dried cells differed from the properties of free spray-dried cells. V (m) for the immobilized enzyme was between 0.08 and 0.09 mM/min, while the V (m) for free enzyme was 0.06-0.07 mM/min. K (m) values differed for immobilized and free spray-dried cells by a factor of between 3 and 5 for (S)-(-)-(alpha)-methylbenzylamine (6.05 mM for immobilized, 1.78 mM for free) and pyruvate (5.0 mM for immobilized, 1.01 mM for free) at 55 degrees C. Optimum pH values were 7.7 and 8.1 for the free spray-dried cells and the immobilized formulation, respectively. The maximum activity for free spray-dried cells was measured at 55 degrees C, whereas for immobilized ones, it was at 60 degrees C. Activation and deactivation energy values for free spray-dried cells were 15.13 and 41.73 kcal/mol, while those for immobilized spray-dried cells were 8.86 and 48.88 kcal/mol, respectively. Overall, as a result of immobilization, an increase in V (m) was measured for the (S)-aminotransferase by 28 to 33% with respect to free enzyme; K (m) increased by a factor of three- to fivefold and had a shift of 5 degrees C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase.

Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying

Spray drying was evaluated as a dehydrating method to preserve the postharvest biocontrol agent Candida sake CPA-1. The effect of drying temperature, carrier, growth and rehydrating medium on the survival of the yeast was studied. Outlet temperature had more influence on the death of the cells than inlet temperature, and survival decreased with increasing temperature. Spray drying at an inlet temperature of 150°C was optimum in terms of viability, powder recovery and moisture content of the product. Use of 10% (v/v) skimmed milk as a carrier gave the highest survival and percentage of powder recovery (34–47%). Rich rehydration media were found to be better than water or phosphate buffer, with slight differences on survival. Spray-dried cells were less effective than fresh ones in controlling Penicillium expansum rot on apples. Spray drying of C. sake was not a good dehydration method as it gave low cell survival, poor recovery of product, and low efficacy.

Survival of Lactobacillus sakei during heating, drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20 g sucrose l(-1) or 12.5 g monosodium glutamate l(-1). No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55 degrees C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%.

Properties of Lactobacillus helveticus CNRZ-32 Attenuated by Spray-Drying, Freeze-Drying, or Freezing

Lactobacillus helveticus CNRZ-32 was attenuated for use in accelerated ripening of Cheddar cheese. The goal was to delay acid production without reducing enzyme activity. Changes in the cellular properties of viability, enzyme activity, lactic acid production, and permeability were measured for each attenuation treatment. The viabilities were highest for frozen and freeze-dried cells. Viabilities were lower for cells spray-dried at an outlet air temperature of 82°C and lowest for 120°C. Aminopeptidase and β-galactosidase activities decreased with each treatment. Low temperature spray-dried cells had the highest residual aminopeptidase and β-galactosidase activities by a substantial margin. Acid production was similar for the frozen, freeze-dried, and low temperature spray-dried cells, and no lag time occurred; however, high temperature spray-dried cells had a 5-h lag time before producing acid. The increase in permeability was greatest for high temperature spray-dried cells. Viability did not necessarily indicate the extent of cell attenuation. Low temperature spray-dried cells had low viability, but the greatest lactic acid production and enzyme activity. High temperature spray-dried cells had the best balance between delaying acid production and maintaining enzyme activity.

Electron Microscopy of Fresh and Dried Cells of Chlorella Pyrenoidosa

Photosynthetic unicellular algae can be cultivated by employing mass production techniques. The harvested Chlorella cells from the mass culturing pools are separated by centrifugation. In the Orient, the pellets are washed and dried in the spray dryer. Finally, the dried powder is packed in vaccum for the starting materials of natural foods. Spray-dried cells of Chlorella were examined by SEM. Most of the droplets of spray-dried cells showed, 50–85μm in diameter (Fig. 1). At the higher magnification, it exhibited that this spherical structure consists of more than a few thousands of single Chlorella cells (Fig. 2), and each cell pilling up or sticking together. The ash-like materials which attached to the surface of cells, probably produced from the cells because of the heat treatment or elevated temperature of hot air from spraying dryers. The cells prepared for SEM by glutaraldehyde fixation and critical point drying had the smooth surface with wrinkled parts. Cells were spherical and measured 2-6 μm in diameter. For the contaminated culture, the photosynthetic purple bacteria are shown to stick on the surface of algal cells. The inside of each particle was revealed by using cryofracture technique. Each particle was occupied by a hole in the center, and its diameter was approximately one-half of the particle in sectional view (Fig. 3).

Conversion of o-succinylbenzoate to dihydroxynaphthoate by extracts of Micrococcus luteus.

Cell-free extracts were prepared from either freshly grown or spray-dried cells of Micrococcus luteus ATCC 4698 by treatment with deoxyribonuclease and lysozyme. These extracts converted o-succinylbenzoic acid (OSB) to 1,4-dihydroxy-2-naphthoic acid (DHNA) as shown by spectrophotofluorometric and radioactivity assays. The conversion required the presence of ATP, CoA, and Mg2+. By use of [2-14C]OSB, the simultaneous production of the spirodilactone form of OSB was also demonstrated. The two products formed from OSB was also demonstrated. The two products formed from OSB were further characterized by gas chromatography combined with mass spectrometry. The production of the spirodilactone was suppressed by the addition of a preparation of the enzyme DHNA synthase obtained from Mycobacterium phlei. (This enzyme catalyzes the conversion of a CoA derivative of OSB to DHNA.) On mild acid treatment, the M. luteus extracts retained the ability to produce spirodilactone but lost the ability to form DHNA. These results are interpreted to mean that an OSB-CoA derivative is an intermediate in the conversion of OSB to DHNA by M. luteus and that two enzymes are involved, one to form the OSB-CoA derivative and the second to carry out a cyclization reaction.