Yves Raymond

Effect of polymers and storage temperature on the stability of freeze-dried lactic acid bacteria

Abstract The effect of the addition of gelatin, xanthan gum or maltodextrins on the survival during freeze-drying and stability during storage of four lactic acid bacteria were studied. The polymers were added to a milk-sucrose base medium and mixed with the cell concentrate. The water activity of the cultures was adjusted to 0.22 prior to storage at −20, 4 or 20 °C. None of the polymers generated marked improvement of the milk-sucrose medium in terms of survival of bacteria during freeze-drying. There were significant differences in mortality rates between strains. Gelatin improved the storage stability of freeze-dried Lactobacillus casei ssp. rhamnosus RO11 and Bifidobacterium longum RO23 cultures kept at 20 and 4 °C, respectively. Also, bacterial populations of Lactococcus lactis ssp. lactis RO58, following 12 months at 4 °C, were higher than controls when gelatin was added to the freeze-drying medium. Overall, the additives had a detrimental effect on the stability of Streptococcus thermophilus RO57 during storage at 20 °C. The B. longum RO23 α- and β-galactosidase activity losses during storage at 20 °C were approximately twice those observed at 4 and −20 °C, while viable count losses at 20 °C were approximately 100× greater than those at 4 °C.

The determination of viable counts in probiotic cultures microencapsulated by spray-coating

An assessment of various methods to determine viable counts (CFU) in freeze-dried and dried microencapsulated (ME) probiotic cultures was carried out. Microencapsulation was done by spray-coating of dried Lactobacillus rhamnosus R0011 or Bifidobacterium longum ATCC 15708 cultures with fat. Rehydration of the ME powders was incomplete when they were added to water and gently agitated. As a result analytical methods based on vortexing of rehydrated ME cultures and which did not incorporate a high-shear homogenization (HSH) step underestimated the viable counts. The CFU of ME cultures were identical when methods using either blender or generator probes high-shear homogenization (HSH) were carried out. Furthermore HSH reduced the variability of the CFU results of both free-cell and ME cultures by a factor of three. The addition of an emulsifier (Tween 80) in the rehydrating medium to dissolve fat did not improve CFU counts when generator probes were used for HSH. The presence of fat in the ME product, or when added to the rehydration medium, improved CFU counts of B. longum but not of L. rhamnosus.

PHYSICAL FACTORS INFLUENCING THE PRODUCTION OF STRAWBERRY AROMA BY PSEUDOMONAS FRAGI GROWN IN SKIM MILK

SummaryThe influence of temperature, agitation speed, pH and biomass on the synthesis of 19 metabolites contributing to a strawberry aroma was followed over a 72 h fermentation of skim milk byPseudomonasfragi. Amongst the major odor-active metabolites were ethyl butyrate, ethyl hexanoate, ethyl 2-hexenoate, ethyl crotonate, ethyl isovalerate and ethyl 2-methyl hexanoate. Up to 17 ppm of some of these metabolites were detected at 60 h of fermentation, approximately 36 h after the beginning of the stationary growth phase. The production of most odor-active metabolites was higher at 11°C and 150 RPM than at 15, 20 or 25°C and 200 or 250 RPM. The development of off-aromas was observed at high temperatures and at high agitation speeds.

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.

ENHANCEMENT OF FRUITY AROMA PRODUCTION OF PSEUDOMONAS FRAGI GROWN ON SKIM MILK, WHEY AND WHEY PERMEATE SUPPLEMENTED WITH C3-C7 FATTY ACIDS

SummaryPseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C3-C7 fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C3-C7 fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.

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 (aw) 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 aw = 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 aw = 0.96. Growing the cells in complex media adjusted at aw = 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.

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.

Production of fatty acid ethyl esters by Pseudomonas fragi under conditions of gas stripping

Abstract Pseudomonas fragi biomass was produced on whey and whey permeate with and without supplementation with various concentrations of butyric or valeric acid. The biomass produced in whey supplemented with 0·1% butyric acid possessed the highest ethylating activity of valeric acid in the presence of ethanol. The performance of the ethylating activity of P. fragi biomass was studied in aqueous medium under conditions of in-situ product removal using gas stripping. From 1000 ppm valeric acid and 2000 ppm ethanol, resting cells of P. fragi produced more than 800 ppm total fatty acid ethyl esters of which valeric acid ethyl ester accounted for more than 90%. The use of a gas stripping technique resulted in a 3.6-fold and 4.4-fold increase in the formation of total fatty acid and valeric acid ethyl esters, respectively, as compared to bioconversions conducted under conditions of product accumulation.

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.

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.