Effect of probiotic bacteria on porcine rotavirus OSU infection of porcine intestinal epithelial IPEC-J2 cells.

Rotavirus infections in nursing or post-weaning piglets are known to cause diarrhea, which can lead to commercial losses. Probiotic supplementation is used as a prophylactic or therapeutic approach to dealing with microbial infections in humans and animals. To evaluate the effect of probiotic bacteria on porcine rotavirus infections, non-transformed porcine intestinal epithelial IPEC-J2 cells were used as an in vitro model, and three different procedures were tested. When cells were exposed to seven probiotics at concentrations of 105, 106, or 107 CFU/mL for 16 h and removed before rotavirus challenge, infection reduction rates determined by flow cytometry were as follows: 15% (106) and 18% (105) for Bifidobacterium longum R0175, 15% (107) and 16% (106) for B. animalis lactis A026, and 15% (105) for Lactobacillus plantarum 299V. When cells were exposed to three selected probiotic strains for 1 h at higher concentrations, that is, 108 and 5 × 108 CFU/mL, before infection with rotavirus, no significant reduction was observed. When the probiotic bacteria were incubated with the virus before cell infection, a significant 14% decrease in the infection rate was observed for B. longum R0175. The results obtained using a cell-probiotics-virus platform combined with flow cytometry analysis suggest that probiotic bacteria can have a protective effect on IPEC-J2 cells before infection and can also prevent rotavirus infection of the cells.

Functional Microbes and Their Incorporation into Foods and Food Supplements: Probiotics and Postbiotics.

Life expectancy has dramatically increased over the past 200 years, but modern life factors such as environmental exposure, antibiotic overuse, C-section deliveries, limited breast-feeding, and diets poor in fibers and microbes could be associated with the rise of noncommunicable diseases such as overweight, obesity, diabetes, food allergies, and colorectal cancer as well as other conditions such as mental disorders. Microbial interventions that range from transplanting a whole undefined microbial community from a healthy gut to an ill one, e.g., so-called fecal microbiota transplantation or vaginal seeding, to the administration of selected well-characterized microbes, either live (probiotics) or not (postbiotics), with efficacy demonstrated in clinical trials, may be effective tools to treat or prevent acute and chronic diseases that humans still face, enhancing the quality of life.

Strategies to improve the survival of probiotic Lacticaseibacillus rhamnosus R0011 during the production and storage of granola bars.

The goal of this study was to evaluate the effectiveness of two approaches to protect the viability of probiotic cells during granola bar manufacturing and storage: microencapsulation (ME) and inclusion in chocolate chips. In the process used, hot honey (138 °C) was blended with cereal ingredients, resulting in an initial blend temperature of 52 °C. Chocolate chips carrying probiotics were added; however, when the blend was cooled to 42 °C. The viability of Lacticaseibacillus rhamnosus R0011 probiotic was assessed by flow cytometry (FC) and plating (CFU). There was an uneven distribution of inoculated probiotic bacteria throughout the cereal bars, resulting in variability in the CFU data. By providing total and viable counts, FC assessed the correct number of inoculated cells in the sample, which enabled the accurate calculation of survival levels. Spray coating with ME increased survival during manufacturing, but ME in alginate particles was detrimental. Including the cultures in chocolate improved the stability of the probiotics during storage at 25 °C, but only in the first 4 weeks. FC analyses showed that viability losses during bar manufacturing could be linked to damage to the cell membrane, but less so during storage.

Effect of the homogenization technique on the enumeration of psychrotrophic bacteria in food absorbent pads.

Four methods were tested for enumerating bacteria present in the absorbent pads (AP) used in packaging chicken and other meats. Viable counts were ascertained at day 0 and day 7 (d0 and d7, respectively). Sampling bacterial cells from AP were carried out using a countertop blender, Stomacher, sonication, and blender in combination to sonication. The release of bacterial cells by breaking down the AP with the blender resulted in the highest CFU counts. At d0, a bacterial recovery rate of 94% was obtained with the blender, while the recovery rates using Stomacher or sonication alone were 58% and 73%, respectively. At d7, the Stomacher treatment also gave the lowest colony forming unit (CFU) values in the AP incubated at 7 °C. Sonication of the AP prior to homogenization with the blender did not increase CFU counts. Results suggested that breaking down the AP with a blender gives higher CFU levels than the Stomacher, which is the most commonly used technique for this purpose.

Microencapsulation of a Staphylococcus phage for concentration and long-term storage.

In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents for the control of foodborne pathogens. The aim of this study was to evaluate microencapsulation (ME) as a tool to concentrate and store staphylococcal bacteriophages. As a proof of concept, phage Team1 belonging to the Myoviridae family was microencapsulated in alginate gel particles of 0.5 mm (micro-beads) and 2 mm (macro-beads) of diameter. Gel contraction occurred during the hardening period in the CaCl2 solution, and the diameters of the initial alginate droplets shrunk by 16% (micro-beads) and 44% (micro-beads). As compared to the phage counts in the alginate solution, this contraction resulted in the increase of the phage titers, per g of alginate gel, by factors of 2 (micro-beads) and 6 (micro-beads). The encapsulation yield was highest in the macro-beads. Although phage Team1 was successfully frozen in beads, ME did not improve phage stability to freeze-drying. The addition of glycerol protected the microencapsulated phages during freezing but had no effect on free phage suspensions. Finally, ME improved storage stability at 4 °C but had no impact on freezing or drying over three months of storage.

Effect of electro-activated sweet whey on growth of Bifidobacterium, Lactobacillus, and Streptococcus strains under model growth conditions.

Recently, we demonstrated the efficacy of electro-activation to improve the functionalities of whey that can be used as a prebiotic and antioxidant agent through lactulose and Maillard reaction products formation. The aim of the present study was to evaluate the effect of electro-activated sweet whey (EA-whey) on growth of probiotics of Bifidobacterium, Lactobacillus, and Streptococcus strains in pure cultures and to compare EA-whey with non-electro-activated whey, lactulose, lactose, sucrose, glucose and galactose at different concentrations (1.25, 2.5 and 5%). The bacterial growth was monitored through maximum optical density (ODmax) and maximum growth rate (µmax) measurements. Moreover, the effects of EA-whey on the growth of L. johnsonii La-1 in the presence of oxygen was assessed. FTIR spectroscopy analyses of the bacterial membrane structure were monitored as a function of EA-whey concentration. The results showed that EA-whey enhanced the growth of all the test bacteria. They clearly demonstrated a promoting bifidogenic effect of EA-whey compared to lactulose. The growth of L. johnsonii La-1 was greatly enhanced under aerobic conditions by the supplementation of the growth medium with EA-whey. This growth promoting effect could be related to the ability of EA-whey to prevent the accumulation of hydrogen peroxide, its high antioxidant capacity and lactulose content. Moreover, FTIR spectra showed that EA-whey acts as an antioxidant in regards to cell membrane lipids oxidation by oxygen species and limited their adverse effect on probiotic bacteria during their growth. Thus, EA-whey, a potential prebiotic and antioxidant, could be used as active ingredient in manufacturing functional fermented dairy products.

The effect of bacteriophages on the acidification of a vegetable juice medium by microencapsulated Lactobacillus plantarum.

Starter cultures are increasingly being used for the production of sauerkraut, kimchi and other fermented vegetables. The goal of this study was to determine whether the microencapsulation of a bacterial culture can prevent phage infection during vegetable fermentation. Lactobacillus plantarum HER1325 was microencapsulated in alginate beads. Some beads were used without further processing, while others were freeze-dried prior to testing. Fresh beads (diameter of 2 mm) and dried cultures of the lactobacilli (particle size of 53-1000 µm) were added to a vegetable juice medium (VJM) at 1 × 107 CFU/mL. The virulent phage HER325 was added at an initial titer of 1 × 104 PFU/mL. In the absence of phages, the pH of the vegetable juice dropped to 4.2 after 40 h of fermentation at 19 °C. In the presence of phage HER325, acidification by both the non-microencapsulated and microencapsulated starter cultures stopped after 24 h. In all assays, the alginate particles dissolved during the 40 h of VJM fermentation. When 15 g/L of calcium chloride was added to the VJM, the alginate beads did not dissolve and significant phage protection was observed. The results suggest that phage-protected microencapsulated starter cultures can be used for vegetable fermentation if means are taken to prevent them from dissolving during acidification.

Electro-activation of sweet defatted whey: Impact on the induced Maillard reaction products and bioactive peptides.

Electro-activation was used to add value to sweet defatted whey. This study aimed to investigate and to characterize the bioactive compounds formed under different electro-activation conditions by molecular and proteomic approaches. The effects of electric current intensity (400, 500 or 600mA) and whey concentration (7, 14 or 21% (w/v)) as a function of the electro-activation time (0, 15, 30 or 45min) were evaluated. The targeted dependent variables were the formation of Maillard reaction products (MRPs), protein hydrolysates and glycated compounds. It was shown that the MRPs derived from electro-activated whey at a concentration of 14% had the highest potential of biological activity. SDS-PAGE analyses indicated the formation of hydrolysates and glycated compounds with different molecular weight distributions. FTIR indicated the predominance of intermediate MRPs, such as the Schiff base compounds. LC-MS/MS and proteomics analysis showed the production of multi-functional bioactive peptides due to the hydrolysis of whey proteins.

Effects of production methods and protective ingredients on the viability of probiotic Lactobacillus rhamnosus R0011 in air-dried alginate beads.

The goal of this study was to use a microencapsulation technology to prepare air-dried concentrated cultures of Lactobacillus rhamnosus R0011. The cultures were microencapsulated in alginate beads, which were added to a growth medium to allow cell multiplication inside the matrix; the beads were recovered, dipped in protective solutions, and air-dried. The effects of fermentation technology and of the composition of the protective solutions on subsequent survival during air-drying were examined. The cells prepared under a constant pH of 6.2 had only 2.5% survival to air-drying at 25 °C when the protective solution was composed of sucrose and phosphate. Allowing the pH to drop to 4.2 during the biomass production step and using a protective medium composed of glycerol, maltodextrin, yeast extract, and ascorbate increased survival to 20%. If the ingredients of the protective medium at the beginning of drying were concentrated at a water activity of 0.96 rather than 0.98, survival during air-drying increased further to 56%. This rate was similar to that of a traditional freeze-drying process. These data suggest that applying a combination of acid and osmotic stresses to L. rhamnosus R0011 cells improves their subsequent stability during the air-drying process. Dried microencapsulated cultures having 2.6 × 1011 CFU·g-1 were obtained.

Contribution to the production of lactulose-rich whey by in situ electro-isomerization of lactose and effect on whey proteins after electro-activation as confirmed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cheese-whey, a major co-product of the dairy industry, has recently been the subject of many technological applications. We studied the bioconversion of whey into valuable bio-products such as a potential lactulose prebiotic and compounds with antioxidant properties. This paper examines efficiency, safety, and economics of electro-activation as an eco-friendly technology for a maximum valorization of whey. Thus, a bottom-up approach was therefore addressed. The effect of 4 experimental parameters--low working temperatures (0, 10, and 25 °C), current intensities (400, 600, and 800 mA), volume conditions (100, 200, and 300 mL), and feed concentrations [7, 14, and 28% (wt/vol)]--on lactose-whey isomerization to lactulose under electro-activation process were studied. Structural characteristics of whey proteins and antioxidant functionality were also investigated. The results showed a compromise to be reached between both parameters. Therefore, the maximum yield of 35% of lactulose was achieved after 40 min of reaction at the working temperature of 10 °C under 400 mA electric current field and 100-mL volume conditions with using feed solution at 7% (wt/vol). The isomerization of lactose to lactulose is accomplished by subsequent degradation to galactose, but only at a very small amount. Additionally, whey electro-activation showed significantly elevated antioxidant capacity compared with the untreated samples. The enhancement of antioxidant functionality of whey electro-activation resulted from the synergistic effect of its partial hydrolysis and the formation of antioxidant components that were able to scavenge free radicals. In conclusion, the findings of this study reveal that the whey treated by the safety electro-activation technology has both lactulose-prebiotic and antioxidant properties and could have a substantial application in the manufacture of pharmaceutical and functional foods.

Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains.

The use of flow cytometry to accurately ascertain total and viable counts of Lactobacillus rhamnosus in chocolate.

The goals of this study were to evaluate the precision and accuracy of flow cytometry (FC) methodologies in the evaluation of populations of probiotic bacteria (Lactobacillus rhamnosus R0011) in two commercial dried forms, and ascertain the challenges in enumerating them in a chocolate matrix. FC analyses of total (FC(T)) and viable (FC(V)) counts in liquid or dried cultures were almost two times more precise (reproducible) than traditional direct microscopic counts (DCM) or colony forming units (CFU). With FC, it was possible to ascertain low levels of dead cells (FC(D)) in fresh cultures, which is not possible with traditional CFU and DMC methodologies. There was no interference of chocolate solids on FC counts of probiotics when inoculation was above 10(7) bacteria per g. Addition of probiotics in chocolate at 40 °C resulted in a 37% loss in viable cells. Blending of the probiotic powder into chocolate was not uniform which raised a concern that the precision of viable counts could suffer. FCT data can serve to identify the correct inoculation level of a sample, and viable counts (FCV or CFU) can subsequently be better interpreted.

Growth-promoting effects of pepsin- and trypsin-treated caseinomacropeptide from bovine milk on probiotics.

Probiotic Lactobacillus and Bifidobacterium species are generally fastidious bacteria and require rich media for propagation. In milk-based media, they grow poorly, and nitrogen supplementation is required to produce high bacterial biomass levels. It has been reported that caseinomacropeptide (CMP), a 7-kDa peptide released from κ-casein during renneting or gastric digestion, exhibits some growth-promoting activity for lactobacilli and bifidobacteria. During the digestive process, peptides derived from CMP are detected in the intestinal lumen The aim of this study was to evaluate the effects of peptic and tryptic digests of CMP on probiotic lactic acid bacteria growth in de Man, Rogosa and Sharpe broth (MRS) and in milk during fermentation at 37 °C under anaerobic conditions. The study showed that pepsin-treated CMP used as supplements at 0.5 g/l can promote the growth of probiotics even in peptone-rich environments such as MRS. The effect was strain-dependent and evident for the strains that grow poorly in MRS, with an improvement of >1.5 times (P<0.05) by addition of pepsin-treated CMP. Trypsin-treated CMP was much less efficient as growth promoter. Moreover, pepsin-treated CMP was effective in promoting the growth in milk of all probiotic lactic acid bacteria tested, with biomass levels being improved significantly, by 1.7 to 2.6 times (P<0.05), depending on the strain. Thus, supplementation of MRS and of milk with pepsin-treated CMP would be advantageous for the production of high biomass levels for Bifidobacteria and Lactobacilli.

Thermostability of Probiotics and Their α -Galactosidases and the Potential for Bean Products.

Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α -galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α -galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α -galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α -galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α -galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans.

Effect of bovine colostrum, cheese whey, and spray-dried porcine plasma on the in vitro growth of probiotic bacteria and Escherichia coli.

The aim of this study is to evaluate the effects of defatted colostrum (Col), defatted decaseinated colostrum whey, cheese whey, and spray-dried porcine plasma (SDPP) as supplements of a growth medium (de Man - Rogosa - Sharpe (MRS) broth) on the multiplication of lactic acid bacteria, probiotic bacteria, and potentially pathogenic Escherichia coli. Using automated spectrophotometry (in vitro system), we evaluated the effect of the 4 supplements on maximum growth rate (µ(max)), lag time (LagT), and biomass (OD(max)) of 12 lactic acid bacteria and probiotic bacteria and of an E. coli culture. Enrichment of MRS broth with a Col concentration of 10 g/L increased the µ(max) of 5 of the 12 strains by up to 55%. Negative effects of Col or SDPP on growth rates were also observed with 3 probiotic strains; in one instance µ(max) was reduced by 40%. The most effective inhibitor of E. coli growth was SDPP, and this effect was not linked to its lysozyme content. The positive effect of enrichment with the dairy-based ingredient might be linked to enrichment in sugars and increased buffering power of the medium. These in vitro data suggest that both Col and SDPP could be considered as supplements to animal feeds to improve intestinal health because of their potential to promote growth of probiotic bacteria and to inhibit growth of pathogenic bacteria such as E. coli.

Dissolution of Lipid-Based Matrices in Simulated Gastrointestinal Solutions to Evaluate Their Potential for the Encapsulation of Bioactive Ingredients for Foods.

The goal of the study was to compare the dissolution of chocolate to other lipid-based matrices suitable for the microencapsulation of bioactive ingredients in simulated gastrointestinal solutions. Particles having approximately 750 µm or 2.5 mm were prepared from the following lipid-based matrices: cocoa butter, fractionated palm kernel oil (FPKO), chocolate, beeswax, carnauba wax, and paraffin. They were added to solutions designed to simulate gastric secretions (GS) or duodenum secretions (DS) at 37°C. Paraffin, carnauba wax, and bees wax did not dissolve in either the GS or DS media. Cocoa butter, FPKO, and chocolate dissolved in the DS medium. Cocoa butter, and to a lesser extent chocolate, also dissolved in the GS medium. With chocolate, dissolution was twice as fast as that with small particles (750 µm) as compared to the larger (2.5 mm) ones. With 750 µm particle sizes, 90% dissolution of chocolate beads was attained after only 60 minutes in the DS medium, while it took 120 minutes for 70% of FPKO beads to dissolve in the same conditions. The data are discussed from the perspective of controlled release in the gastrointestinal tract of encapsulated ingredients (minerals, oils, probiotic bacteria, enzymes, vitamins, and peptides) used in the development of functional foods.

Encapsulation of coenzyme Q10 in a simple emulsion-based nutraceutical formulation and application in cheese manufacturing.

Coenzyme Q10 (CoQ10) was encapsulated successfully in a nutraceutical formulation composed of calcium caseinate, flaxseed oil and lecithin. The effect of CoQ10 on the physico-chemical stability of emulsions was compared to emulsions without CoQ10. According to ATR-FTIR analysis, emulsions were found to be more stable in the presence of CoQ10. The emulsion with CoQ10 was used as a functional cream in the cheese making process. The retention rate of CoQ10, composition and cheese yield were also determined. Quantification of CoQ10 by HPLC showed that the retention of this lipophilic agent into cheese matrix was 93% and equivalent to the total lipid retention. Protein retention and cheese yield were not affected by the addition of the functional cream. For the first time, CoQ10 has been encapsulated in a cheese matrix, hence demonstrating that CoQ10 could be used in the development of functional cheeses.

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures.

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a (w)) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a (w) = 0.96 or 0.99 were then recovered by centrifugation, added to a sucrose-phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a (w) = 0.96. Growing the cells in complex media adjusted at a (w) = 0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose-phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.

Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices.

Due to the fact that probiotic cells need to be alive when they are consumed, culture-based analysis (plate count) is critical in ascertaining the quality (numbers of viable cells) of probiotic products. Since probiotic cells are typically stressed, due to various factors related to their production, processing and formulation, the standard methodology for total plate counts tends to underestimate the cell numbers of these products. Furthermore, products such as microencapsulated cultures require modifications in the release and sampling procedure in order to correctly estimate viable counts. This review examines the enumeration of probiotic bacteria in the following commercial products: powders, microencapsulated cultures, frozen concentrates, capsules, foods and beverages. The parameters which are specifically examined include: sample preparation (rehydration, thawing), dilutions (homogenization, media) and plating (media, incubation) procedures. Recommendations are provided for each of these analytical steps to improve the accuracy of the analysis. Although the recommendations specifically target the analysis of probiotics, many will apply to the analysis of commercial lactic starter cultures used in food fermentations as well.