Freeze-dried Bacteria Research Articles

Probiotic Bacteria Survival and Shelf Life of High Fibre Plant Snack - Model Study

The study aimed to develop plant-based model snacks that are high in fibre, contain probiotic bacteria and are convenient for long-term storage. The research focused on selecting a suitable form of probiotic bacteria (active biomass, microencapsulated, freeze-dried), inoculation method (in the base mass or in the filling of a snack) and appropriate storage conditions (4°Cor 20 °C). The potential synbiotic properties were evaluated. The microencapsulated bacteria had the highest survival rate at 4 °C, while the freeze-dried bacteria showed better survival rates at 20 °C. Probiotics had a higher survival rate when enclosed inside snacks with a low water activity (aw = 0.27) peanut butter filling than in snacks without filling (aw = 0.53). Enclosing the probiotics in a low aw filling ensures their survival at ambient temperature for 5 months at a count higher than 6 log CFU/g. The snacks exhibited high antioxidant capacity (average 300 mg ascorbic acid equivalent/100 g), polyphenol content (average 357 mg gallic acid equivalent/100 g) and high fibre content (average 10.2 g/100 g). The sensory analysis showed a high overall quality of the snacks (average 7.1/10 of the conventional units). Furthermore, after six months of storage, significant changes were observed in the antioxidant properties, polyphenol content and texture of the snacks, while their sensory quality remained unchanged. Moreover, a potential synbiotic effect was observed. The method used to assess bacterial growth indicated significantly higher values in the model snacks compared to a control sample. Therefore, this study has effectively addressed the gap in knowledge regarding the survival of probiotics in snacks of this nature.

Dynamics of Active Fluorescent Units (AFU) and Water Activity (aw) Changes in Probiotic Products-Pilot Study.

The flow cytometry method (FCM) is a widely renowned practice increasingly used to assess the microbial viability of probiotic products. Additionally, the measurement of water activity (aw) can be used to confirm the presence of viable cells in probiotic products throughout their shelf lives. The aim of this study was to investigate the correlation between changes in aw and variations in active fluorescent units (AFU), a unit commonly used in flow cytometry method, during the aging of probiotic products containing freeze-dried bacteria. We controlled the stability of probiotic products for bacterial counts (using ISO 19344 method) and aw levels in commercially available capsules containing freeze-dried bacteria such as Lactobacillus sp. or combinations of Lactobacillus sp. and Bifidobacterium sp. in standard conditions (25 ± 2 °C and 60% relative humidity) over a period of 24 months. During this time, the bacterial contents decreased by 0.12 Log10 in the single-strain product, by 0.16 Log10 in the two-strain product and by 0.26 Log10 in the multi-strain product. With the increase in aw, the number of bacteria decreased but the aw at the end point of the stability study did not exceed 0.15 in each of the three tested products. FCM combined with aw is a prospective analysis that can be used to assess the stability of probiotic products, both for its ability to detect bacterial viability and for practical (analysis time) and economic reasons.

Enhanced heat tolerance of freeze-dried Enterococcus faecium NRRL B-2354 as valid Salmonella surrogate in low-moisture foods

In microbial studies of low-moisture foods (LMFs, water activity less than 0.85), freeze-dried bacteria benefit us to inoculate LMFs without introducing extra water or altering food physiochemical properties. However, the freeze-drying process would bring unavoidable damage to bacterial cells and results in less-resistant inoculum that are unlikely to be qualified in microbial studies. Herein, we enhanced bacterial heat tolerance by subjecting the cells to mild heat (42-50 °C) to counteract the reduced heat tolerance and survivability of freeze-dried bacteria. Enterococcus faecium NRRL B-2354 (E. faecium), a Salmonella surrogate in LMFs, was used as the target microorganism because it was widely accepted in microbial validation of thermal pasteurizing LMFs. Three types of LMFs (peanut powder, protein powder, and onion powder) were used as LMFs models to validate the freeze-dried E. faecium in comparison with Salmonella enterica Enteritidis PT 30 (S. Enteritidis) prepared by the traditional aqueous method. The heat tolerance (D65℃ value) of E. faecium increased at all treatments and peaked (+31.48 ± 0.13%) at temperature-time combinations of 45 °C-60 min and 50 °C-5 min. Survivability of freeze-dried inoculum and its heat tolerance retained well within 50 d storage. The freeze-dried E. faecium was prepared in this study brought equal or higher heat tolerance (D85℃ or D75℃) than S. Enteritidis in tested LMFs models. For instance, the D85℃ of freeze-dried E. faecium (heat-treated at 50 °C for 5 min) and S. Enteritidis in whole egg powder are 35.56 ± 1.52 min and 28.41 ± 0.41 min, respectively. The freeze-dried E. faecium with enhanced heat tolerance appears to be a suitable Salmonella surrogate for dry-inoculating LMFs. Our protocol also enables industry-scale production of freeze-dried inoculum by broth-cultivation method combined with mild-heat treatment.

Effect of protective agents on the storage stability of freeze-dried Ligilactobacillus salivarius CECT5713.

Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits but remains commercially underexploited due to its inability to survive during long-term storage in the dried state. Our objective was to study the effect of various protective molecules (maltodextrin, trehalose, antioxidants, and fructooligosaccharides), being efficient on other bacteria, on the freeze-dried stability of L. salivarius CECT5713. The culturability was evaluated after freezing, freeze-drying, and subsequent storage at 37°C, as well as the biochemical composition of cells in an aqueous environment using Fourier transform infrared (FTIR) micro-spectroscopy. The assignment of principal absorption bands to cellular components was performed using data from the literature on bacteria. The membrane fatty acid composition was determined after freeze-drying and storage. Glass transition temperature of the liquid and freeze-dried bacterial suspensions and water activity of the freeze-dried samples were measured. The best storage stability was observed for the formulations involving maltodextrin and antioxidants. The analysis of the FTIR spectra of freeze-thawed cells and rehydrated cells after freeze-drying and storage revealed that freeze-drying induced damage to proteins, peptidoglycans of the cell wall and nucleic acids. Storage stability appeared to be dependent on the ability of the protective molecules to limit damage during freeze-drying. The inactivation rates of bacteria during storage were analyzed as a function of the temperature difference between the product temperature during sublimation or during storage and the glass transition temperature, allowing a better insight into the stabilization mechanisms of freeze-dried bacteria. Maintaining during the process a product temperature well below the glass transition temperature, especially during storage, appeared essential for L. salivarius CECT5713 storage stability. KEY POINTS: • L. salivarius CECT5713 highly resisted freezing but was sensitive to freeze-drying and storage. • Freeze-drying and storage mainly altered cell proteins, peptidoglycans, and nucleic acids. • A glassy matrix containing maltodextrin and an antioxidant ensured the highest storage stability.

Preservation of Paenibacillus polymyxa BTK01 and Bacillus subtilis BTK07 as lignocellulolytic bacterial starters for industrial applications: Physicochemical conditions, enzyme stability, freeze-drying processes and cryoprotection

In a previous study, Paenibacillus polymyxa BTK01 (BTK01) and Bacillus subtilis BTK07 (BTK07) were identified as lignocellulolytic strains that produced xylanase (XLN) and mannanase (MNN), respectively. As a biocontrol agent and a biofertilizer, these bacteria can be beneficial for organic agriculture, and they could be developed by upscaling as a bacterial starter. Moreover, the enzymes produced can be useful in the wood pulp industry. Therefore, this study focused on determining the physicochemical conditions for enzyme catalysis and for preserving BTK01 and BTK07 using a freeze-drying process. We show that the optimum physicochemical conditions for XLN and MNN produced by these strains were clearly defined by pH 7–8 and 40–60 °C, in which the lignocellulolytic enzymes of BTK01 were thermally stable. The XLN from BTK01 was stable at 40 °C but became unstable at lower temperatures. As expected, freeze-drying without cryoprotectant reduced bacterial survival rather than enzymatic activity, but the 30-day-storage of the freeze-dried bacteria primarily affected enzyme catalysis. For added value, the fermentation broth could be divided into sediment and supernatant to obtain freeze-dried products as a bacterial starter and a crude enzyme, respectively. Additionally, formulating skimmed milk and glycerol as a cryoprotectants could improve bacterial survival and enzymatic activity of freeze-dried bacteria, particularly BTK07. Accordingly, the results indicate that the freeze-dried products of BTK01 and BTK07 are suitable for industrial-scale production to supply the agricultural sector and other related fields.

Effect of Protectants Made From Sucrose and Antioxidant Blends On The Stability of Freeze-Dried Lactic Acid Bacteria

Effect of Protectants Made From Sucrose and Antioxidant Blends On The Stability of Freeze-Dried Lactic Acid Bacteria

Water sorption characteristics of freeze-dried bacteria in low-moisture foods

Water sorption isotherms of bacteria reflect the water activity with the change of moisture content of bacteria at a specific temperature. The temperature-dependency of water activity change can help to understand the thermal resistance of bacteria during a thermal process. Thermal resistance of bacteria in low-moisture foods may differ significantly depending on the physiological characteristics of microorganisms, including cell structure, existence of biofilms, and growth state. Previous studies demonstrated that the incremental change of aw in bacterial cells during thermal treatments resulted in changes in their thermotolerance. In this study, a pathogen associated with low-moisture foods outbreaks, Salmonella Enteritidis PT30 (in planktonic and biofilm forms), and its validated surrogate, Enterococcus faecium, were lyophilized and their water sorption isotherms (WSI) at 20, 40, and 60 °C were determined by using a vapor sorption analyzer and simulated by the Guggenheim, Anderson and De Boer model (GAB). The published thermal death times at 80 °C (D80 °C-values) of these bacteria in low-moisture environments were related with their WSI-derived aw changes. The results showed that planktonic E. faecium and biofilms of Salmonella, exhibiting higher thermal resistance compared to the planktonic cultures of Salmonella, had a smaller increase in aw when thermally treated from 20 to 60 °C in sealed test cells. The computational modeling also showed that when temperature increased from 20 to 60 °C, with an increase in relative humidity from 10% to 60%, freeze-dried planktonic E. faecium and Salmonella cells would equilibrate to their surrounding environments in 0.15 s and 0.25 s, respectively, suggesting a rapid equilibration of bacterial cells to their microenvironment. However, control of bacteria with different cell structure and growth state would require further attentions on process design adjustment because of their different water sorption characteristics.

Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage

BackgroundStabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and maintaining a low aw during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C.ResultsThe addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05).ConclusionsThe addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.

Cytotoxic effects of dental calculus particles and freeze-dried Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum on HSC-2 oral epithelial cells and THP-1 macrophages.

Periodontitis is an inflammatory disease initiated by dental deposits. Microorganisms in the dental biofilm induce cell death in epithelial cells, contributing to the breakdown of epithelial barrier function. Recently, dental calculus has also been implicated in pyroptotic cell death in oral epithelium. We analyzed the cytotoxic effects of dental calculus and freeze-dried periodontopathic bacteria on oral epithelial cells and macrophages. HSC-2 (human oral squamous carcinoma cells) and phorbol 12-myristate 13-acetate-differentiated THP-1 macrophages were exposed to dental calculus or one of two species of freeze-dried bacterium, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. Following incubation for 24 hours, we measured cytotoxicity via lactate dehydrogenase release. Cells were then incubated with glyburide, an NLRP3 inflammasome inhibitor, to assess the potential role of pyroptosis. We also conducted a permeability assay to analyze the effects on epithelial barrier function. Dental calculus induced dose-dependent cell death in HSC-2 cells, whereas cell death induced by freeze-dried bacteria was insignificant. Conversely, freeze-dried bacteria induced more cell death than dental calculus in THP-1 macrophages. Cell death induced by dental calculus but not by freeze-dried bacteria was inhibited by glyburide, indicating that these are different types of cell death. In the permeability assays, dental calculus but not freeze-dried bacteria attenuated the barrier function of HSC-2 cell monolayers. Due to the low sensitivity of HSC-2 cells to microbial cytotoxicity, dental calculus had stronger cytotoxic effects on HSC-2 cell monolayers than freeze-dried A. actinomycetemcomitans and F. nucleatum, suggesting that it plays a critical role in the breakdown of crevicular/pocket epithelium integrity.

Probiotic Lactobacilli Administration Induces Changes in the Fecal Microbiota of Preweaned Dairy Calves.

Early microbial colonization is a determinant factor in animal health, and probiotic administration has been demonstrated to modulate intestinal microbiota and promote health in dairy calves. The objective of this study was to evaluate changes in calves' fecal microbiota after the administration of two probiotic lactobacilli strains that had previously exhibited beneficial effects in calves' health in relation to neonatal calf diarrhea. An in vivo assay was performed with 30 newborn male Holstein calves that were divided into three groups. Two groups were orally administered with two different lactobacilli strains (Lactobacillus johnsonii TP1.6 or Limosilactobacillus reuteri TP1.3B), and the third was the control group. Calves (5 to 9days old) were administered with freeze-dried bacteria once a day for 10 consecutive days. Feces samples were taken before the first administration (day 0) and then again on days 10 and 21, and the V4 region of the bacterial 16S ribosomal gene was sequenced with an Illumina MiSeq 250 paired-end platform. The administration of both strains significantly affected the total bacterial community composition, and the effect lasted for 11days after the last dose. In particular, amplicon sequence variants related to Bifidobacterium and Akkermansia genera were significantly higher in both treated groups. Therefore, modulation of the intestinal microbiota is a potential mechanism of action behind the beneficial effects of these probiotic strains.

Multiparameter flow cytometric enumeration of probiotic-containing commercial powders

Flow cytometry (FCM) was used for the assessment of viability and number of freeze-dried bacteria such as: i) Lactobacillus sp.; ii) Lactobacillus sp., Bifidobacterium sp.; iii) and Lactobacillus sp., Bifidobacterium sp. and Lactococcus lactis) in commercially available capsules. Samples were analyzed based on two different physiological parameters: membrane integrity and measurement of the activity of cellular oxido-reductases. The total amount of viable bacteria from each probiotic product varied between 70 and 90%, while dead cells were between 10 and 30%. For product containing Lactobacillus sp. only, the number of bacteria assessed by FCM was 1.2 × 1011 AFU/g (active fluorescent units) compared to plate count (PC) method - 1.4 × 1011 CFU/g. For product containing Lactobacillus sp. and Bifidobacterium sp. the difference between CFU/g and AFU/g was 7.3 × 1010 and 1.3 × 1010 for product containing Lactobacillus sp., Bifidobacterium sp. and Lactococcus lactis. A method based on the enzymatic activity of the cells for product containing Lactobacillus sp. gave an average result: 1.4 × 1011 AFU/g, for product containing Lactobacillus sp., Bifidobacterium sp.: 7 × 1010 AFU/g and for product containing Lactobacillus sp., Bifidobacterium sp. and Lactococcus lactis: 3.7 × 1010 AFU/g. FCM allowed to get results after 2 h compared to PC method where result was obtained after 96 h. Moreover no significant differences were found for AFU/g obtained using the two protocols.

Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria

Bacteria show distinct and characteristic fatty acid (FA) patterns which can be modified by environmental conditions. In this study, we cultivated six plant-pathogenic bacteria of agricultural concern and performed a detailed analysis of the fatty acid composition. The study covered four strains of the gram-negative Xanthomonas campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax citrulli (Ac) and Pseudomonas syringae pv. tomato (Pst), and two strains of the gram-positive Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies (Ssc). After cultivation, freeze-dried bacteria samples were transesterified and analysed by gas chromatography with mass spectrometry in full scan and selected ion monitoring (SIM) modes. Altogether, 44 different FAs were detected in the six strains with individual contributions of 0.01–43.8% to the total FAs. The variety in the six strains ranged between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is typical for most bacteria. In contrast to this, Ac and Pst showed only saturated and monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported before in the literature. Differences were observed in the case of Cmm where many more FAs were detected in the present study. In addition, to the best of our knowledge, the FA pattern of Xp was presented for the first time.

Portable, Disposable, Paper-Based Microbial Fuel Cell Sensor Utilizing Freeze-Dried Bacteria for In Situ Water Quality Monitoring.

Water quality monitoring is becoming an essential part of our lives as increasing human activities continue to spill unknown and unexpected contaminants into our water systems. To ensure the provision of safe and clean water to the public and the ecosystem, the development of rapid and sensitive in situ early warning systems for water toxicity monitoring is crucial. In this work, an entirely paper-based microbial fuel cell sensor utilizing freeze-dried bacteria is demonstrated as a portable and disposable water toxicity sensor. The bacterial cells were preinoculated on the anode reservoir of the device, and they were freeze-dried, making their on-site and on-demand applications possible. Upon rehydration of the bacteria with the water samples, current readings were obtained, and inhibition ratios (IRs) were calculated for different concentrations of formaldehyde as a model toxin. For 0.001, 0.01, and 0.02% of formaldehyde, IRs of 7.88, 16.08, and 23.14% were obtained, respectively. These IRs showed a very good linearity with the formaldehyde concentrations at R2 = 0.995. Additionally, the shelf life of the freeze-dried microbial fuel cell sensor was investigated. Even after 14 days of storage in the desiccator, at 4, and at −20 °C, the performance outputs compared to the new device were all at 96%.

Patterns of Heavy Metals in a Continuous Toxicity Monitoring System using Bioluminescent Bacteria

Objectives:The purpose of this study is to evaluate the effects of heavy metals (Zn<sup>2+</sup>, Pb<sup>2+</sup>, Hg<sup>2+</sup>, Cd<sup>2+</sup>) toxicity in a real-time biosensor which is based on measuring the attenuation of light intensity emitted by <i>Vibrio fischeri</i>.Methods:The inhibition test included four heavy metals and the spiked effluent wastewater. The toxicity of the test samples was assessed by freeze-dried bacteria (<i>Vibrio fischeri</i> NRRL-B-11177). The pH of the samples was adjusted to 7±0.2 before conducting the toxicity tests in oder to eliminate potential pH effects. The results obtained from the real-time biosensor that can be measuring the toxicity of the samples as a function of the emitted light. within a short period time (2 min). And then EC20 and EC50 values was estimated from inhibition curves.Results and Discussion:In case of the inhibition test for dilluents of heavy metals, EC<sub>20</sub> values were estimated as < 0.2 mg/L for Zn(II), 0.45 mg/L for Hg(II), 0.58 mg/L for Cd(II) and 1.95 mg/L for Pb(II) and EC<sub>50</sub> values were estimated as 0.25 mg/L for Zn(II), 0.5~1.0 mg/L for Hg(II), 1.38 mg/L for Cd(II) and 3.76 mg/L for Pb(II). The sensitivity ranking of heavy metals was in order as Zn(II) > Hg(II), > Cd(II) > Pb(II). In case of the inhibition test for the spiked effluent wastewater, EC<sub>20</sub> values were estimated as 0.38 mg/L for Hg(II), 0.58 mg/L for Zn(II), 1.45 mg/L for Pb(II) and 1.95 mg/L for Cd(II) and EC<sub>50</sub> values were estimated as 0.53 mg/L for Hg(II), 1.13 mg/L for Zn(II), 6.44 mg/L for Pb(II) and 7.82 mg/L for Cd(II). The sensitivity ranking of the spiked effluent wastewater was in order as Hg(II) > Zn(II) > Pb(II) > Cd(II). Compared to the Emssion Limit Value (ELV) in Korea, the EC<sub>20</sub> value of Zn(II) was found to be lower than ELV so that it was suitable for screening of Zn(II) as to meet the criteria of effluent wastewater. However, the other EC<sub>20</sub> value of heavy metals can be used to control the unit processes and avoid accidental discharge.Conclusions:The assessment of the toxicity of four heavy metals and spiked samples with them was performed in this study, by real-time biosensor using bioluminescent bacteria. The EC20 value of the spiked sample with Zn(II) has been found to be 0.58 mg/L lower than its ELV so that it was considered as a suitable screening for determining whether or not to exceed the permissible limit value. The use of real-time biosensor is required in combination with physicochemical analyses for an integrated screening of the industrial effluent properties.

Probing the stability and magnetic properties of magnetosome chains in freeze-dried magnetotactic bacteria.

Magnetospirillum gryphiswaldense biosynthesize high-quality magnetite nanoparticles, called magnetosomes, and arrange them into a chain that behaves like a magnetic compass. Here we perform magnetometry and polarized small-angle neutron scattering (SANS) experiments on a powder of freeze-dried and immobilized M. gryphiswaldense. We confirm that the individual magnetosomes are single-domain nanoparticles and that an alignment of the particle moments along the magnetic field direction occurs exclusively by an internal, coherent rotation. Our magnetometry results of the bacteria powder indicate an absence of dipolar interactions between the particle chains and a dominant uniaxial magnetic anisotropy. Finally, we can verify by SANS that the chain structure within the immobilized, freeze-dried bacteria is preserved also after application of large magnetic fields up to 1 T.

Life cycle assessment of the production of stabilized lactic acid bacteria for the environmentally-friendly preservation of living cells

Abstract Among food ingredients, concentrates of lactic acid bacteria are widely used as starters for producing cheeses, fermented milks, meats, vegetables and beneficial health products (probiotics). The first objective of this study was to identify environmental hotspots of the production system of lactic acid bacteria concentrates. The second was to compare the impacts of the two scenarios of stabilization (freezing vs. freeze-drying). To fulfill these objectives, Life Cycle Assessment was performed. Since production processes damage lactic acid bacteria, the impact scores were weighted by the quality of the bacteria, i.e., their physiological state, to obtain meaningful comparisons. Storage and fermentation were the hotspots when bacteria were stabilized by freezing, whereas fermentation and freeze-drying were the hotspots when bacteria were stabilized by freeze-drying. By comparing different storage devices (laboratory vs. walk-in freezers), it was revealed that the device scale was a critical choice when storage was a hotspot. Surprisingly, no benefit was obtained by increasing the storage temperature of freeze-dried products from −20 °C to ambient temperature due to the high bacteria quality loss in such storage conditions. The scenario comparison (frozen vs. freeze-dried bacteria) highlighted the dependence of the results on storage time. The most eco-friendly scenario was frozen bacteria for storage periods of less than 2 months, whereas it was freeze-dried bacteria for storage periods of more than 7 months. Between 2 and 7 months, the conclusion depended on the indicators considered. This study demonstrated improvement options for the processes, including the energy efficiency of freeze-drying and the efficient use of steam and water, as well as optimized cleaning procedures for fermentation. It also highlighted the environmental benefit that could be obtained if the quality of the bacteria could be more efficiently preserved during freeze-drying and storage. This study provides valuable information concerning the preservation of microorganisms and the use of the cold chain.

Okara: A Nutritionally Valuable By-product Able to Stabilize Lactobacillus plantarum during Freeze-drying, Spray-drying, and Storage.

Okara is a nutritionally valuable by-product produced in large quantities as result of soymilk elaboration. This work proposes its use as both culture and dehydration medium during freeze-drying, spray-drying, and storage of Lactobacillus plantarum CIDCA 83114. Whole and defatted okara were employed as culture media for L. plantarum CIDCA 83114. The growth kinetics were followed by plate counting and compared with those of bacteria grown in MRS broth (control). No significant differences in plate counting were observed in the three media. The fatty acid composition of bacteria grown in whole and defatted okara showed a noticeable increase in the unsaturated/saturated (U/S) fatty acid ratio, with regard to bacteria grown in MRS. This change was mainly due to the increase in polyunsaturated fatty acids, namely C18:2. For dehydration assays, cultures in the stationary phase were neutralized and freeze-dried (with or without the addition of 250 mM sucrose) or spray-dried. Bacteria were plate counted immediately after freeze-drying or spray-drying and during storage at 4°C for 90 days. Freeze-drying in whole okara conducted to the highest bacterial recovery. Regarding storage, spray-dried bacteria previously grown in whole and defatted okara showed higher plate counts than those grown in MRS. On the contrary, freeze-dried bacteria previously grown in all the three culture media were those with the lowest plate counts. The addition of sucrose to the dehydration media improved their recovery. The higher recovery of microorganisms grown in okara after freeze-drying and spray-drying processes and during storage was ascribed to both the presence of fiber and proteins in the dehydration media, and the increase in U/S fatty acids ratio in bacterial membranes. The obtained results support for the first time the use of okara as an innovative matrix to deliver L. plantarum. Considering that okara is an agro-waste obtained in large quantities, these results represent an innovative strategy to add it value, providing a symbiotic ingredient with promising industrial applications in the development of novel functional foods and feeds.

Design of a beneficial product for newborn calves by combining Lactobacilli, minerals, and vitamins

ABSTRACTDiarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell’s recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.

Persistence of bifidobacteria in the intestines of calves after administration in freeze-dried form or in fermented milk

In order to improve the gut microbiome of calves, probiotic bacteria can be fed as active living-cells (fermented milk), or as live but inactive (freeze-dried) cultures. Ten bifidobacterial strains with suitable probiotic properties (as determined in our previous study) were tested for survival during the freeze-drying process, and screened for their ability to ferment cow's milk. The viability of both freeze-dried and live-cell cultures during storage was also tested. All of the strains tested were able to ferment cow's milk, with average counts of 8.26 ± 0.62 log CFU/ml. Eight out of the ten strains were able to survive in milk for 2 months in counts higher than 106 CFU/ml. Bifidobacteria showed high viability following the freeze-drying process, with average numbers of 9.03 ± 0.22 log CFU/vial and did not decrease after 12 months of storage. The mixture of rifampicin-resistant variants of bifidobacteria (RRBs) was fed to 2-day-old dairy Charolais calves in the form of living-cells, or as freeze-dried bacteria. The control group was given no probiotics. Survival of the RRBs administered and the numbers of other bacterial groups in faecal samples was monitored by culturing. Bifidobacteria that were administered passed successfully through the upper parts of the gastrointestinal tract, and were found in numbers higher than 109 CFU/g for two weeks. RRBs colonized the intestines of calves for at least 63 days in both treatment groups. Significantly higher total counts of bifidobacteria were found in the treated groups, compared to the control group. Reduction in Escherichia coli and total coliforms numbers, and an increase in lactobacilli counts were observed in both experimental groups following the application of the probiotic mixtures. Our results show that both forms of administering probiotic bifidobacteria to calves are effective, but that the freeze-dried form is more suitable from a practical viewpoint.

Optimization of electrospraying conditions for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion

Electrospraying has recently emerged as a novel microencapsulation technique with potential for the protection of probiotics. However, research efforts are still needed to minimize the viability loss observed during the processing of sensitive strains, and to maximize productivity. The aim of the present work was the optimization of the electrospraying conditions for the microencapsulation of a model probiotic microorganism, Lactobacillus plantarum, within a whey protein concentrate matrix. In a pre-optimization step, the convenience of encapsulating fresh culture instead of freeze-dried bacteria was established. Additionally, a surface response methodology was used to study the effect of the applied voltage, surfactant concentration, and addition of a prebiotic to the formulation on cell viability and productivity. Viability losses lower than 1 log10 CFU were achieved and the bacterial counts of the final products exceeded 8.5 log10 CFU/g. The protection ability of the developed structures during storage and in-vitro digestion was also evaluated.