Claude P. Champagne

Challenges in the Addition of Probiotic Cultures to Foods

Abstract Probiotic cultures are increasingly being added to foods in order to develop products with health-promoting properties. Although the literature is abundant on the beneficial effects of bifidobacteria and Lactobacillus acidophilus on health, little information is available on the challenges industry faces in adding these probiotic cultures to food products. The aim of this article is to examine seven issues that should be addressed when developing functional foods: 1) type or form of probiotic that should be used; 2) addition level required to have a beneficial effect; 3) toxicity; 4) effect of the processing steps on viability; 5) determination, in the product, of the cell populations added; 6) stability during storage; 7) changes in sensory properties of the foods.

Selection and characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and onion vegetable mixtures

An evaluation of various lactic acid bacteria (LAB) for the fermentation of cabbage, carrot and beet-based vegetable products was carried out. As part of a screening process, the growth of 15 cultures in a vegetable juice medium (VJM) was characterized by automated spectrophotometry. Acidification patterns as well as viability during storage of the LAB were also established. There were greater differences between the pure cultures than the mixed ones with respect to growth in VJM and viability during storage. Reductions in viable cell counts during storage of the fermented VJM occurred more rapidly with a Leuconostoc strain than for pediococci or lactobacilli. Inoculation of vegetables was carried out with cultures of Lactobacillus plantarum NK-312, Pediococcus acidilactici AFERM 772 and Leuconostoc mesenteroides BLAC which were rehydrated in a brine. This rehydration procedure was not detrimental to viability. During fermentation of a carrot/cabbage vegetable mix, sugar metabolism was characterized by the assimilation of both glucose and fructose, but sugars remained in the fermented vegetables when acidification stopped. The pH in the LAB-inoculated vegetables after 72 h at 20 degrees C was significantly lower (by 0.2 units) than the uninoculated control. Inoculation with LAB designed for silage fermentation resulted in the inhibition of acetic acid production, and reduced the production of ethanol during fermentation. The selection process on VJM enabled the preparation of a mixed culture that was more rapid than the silage inoculants in acidifying the medium and was more effective in reducing the production of gas during the fermentation and storage of the fermented vegetables.

Antibacterial and antiviral activity of camel milk protective proteins

Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.

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.

Immobilized Cell Technologies for the Dairy Industry

The potential applications of immobilized cell technology (ICT) to the dairy industry are examined. Immobilization modifies the physiology of cells, and the consequences of ICT on lactose as well as citrate metabolism are reviewed. Immobilization also affects the sensitivity of lactic acid bacteria (LAB) to salt and penicillin. ICT can be used to produce starters for the dairy industry, and aspects of biomass production in beads, continuous cell release from beads, and continuous fermentations with filtration cell recycle are examined. Potential applications of ICT to the dairy industry include acidification of raw milk prior to ultrafiltration, inhibition of psychrotrophic bacteria in raw milk, yogurt production, cheese manufacture, and cream fermentations. Impacts of yeast, bacterial, or bacteriophage contaminations in ICT processes as well as their control are discussed.

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.

Microentrapment of probiotic bacteria in a Ca2 +-induced whey protein gel and effects on their viability in a dynamic gastro-intestinal model

Entrapping probiotic bacteria in gels with ionic cross-linking is typically achieved with polysaccharides (alginate, pectin, carraghenan). In this study, whey proteins were used for this purpose by carrying out the Ca2+-induced gelation of pre-heated whey protein isolate (WPI). A Lactobacillus rhamnosus cell suspension was added in a denatured WPI solution in a 30 : 70 volume ratio. Gelation was carried out by extrusion of the cell suspension in a CaCl2 solution. Beads of ∼3 mm diameter were formed. The population in the beads was 8.0 × 108 cells g−1. Entrapment efficiency in gel beads was 96%, with a survival level of 23%. Scanning electron microscopy of beads before freeze-drying showed a tight protein network containing encapsulated Lb. rhamnosus cells homogeneously distributed throughout the matrix. The survival to freeze-drying of the bead-entrapped cells was 41%. Viability of microentrapped cells in a dynamic gastro-intestinal (GI) model was studied and the results were compared to free cells freeze-dried in a milk-based cryoprotective solution, as well as in a pre-denatured WPI solution. Results showed that protein gelation provided protection against acidic conditions in the stomach after 90 min, as well as against bile after 30, 60 and 90 min in the duodenum. Moreover, the milk-based cryoprotective solution was equally effective after 90 min in the duodenum. It is concluded that the gelation of whey proteins induced by Ca2+ ions can protect the cells against adverse conditions of the GI system. However, certain stages in the entrapment process, particularly extrusion in the solution of CaCl2, still need to be optimized in order to reduce the mortality of the cells during gelation.

Antimicrobial Action of Hydrolyzed Chitosan against Spoilage Yeasts and Lactic Acid Bacteria of Fermented Vegetables

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weight chitosan-lactate DP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel's milk

Abstract Electrophoretic SDS-PAGE analysis of purified camel and bovine milk and blood serum proteins revealed different migration patterns and molecular weights. Camel blood serum was fractionated by precipitation with saturated ammonium sulphate (SAS); the optimal recovery of immunoglobulins was at a concentration of 40% x 2 SAS. Isolation of bovine immunoglobulins, using an anion exchange resin such as DEAE-Sephacel was easier than for camel milk and serum since the difference in charge between the two subclasses of bovine immunoproteins IgG1 and IgG2 was greater. Protein-A chromatography was necessary as a first step followed by DEAE-Sephacel, for the purification of camel milk IgG1 and IgG2. Camel blood serum or milk IgG had high affinity to protein-A. However, secretory sIgA and IgM did not bind. Isolation of camel milk lysozyme, lactoferrin and lactoperoxidase was carried out using a carboxy methyl-cellex cation exchange resin. Both camel milk lysozyme and lactoperoxidase were eluted at lower salt molarity than bovine proteins. Camel milk lactoferrin was eluted at higher molarity than bovine milk lactoferrin. Molecular weights of purified camel milk lysozyme, lactoferrin and lactoperoxidase were estimated at 14.4, 79.5 and 78 KDa, respectively; for bovine milk, corresponding values were 14.4, 76 and 72.5 KDa respectively. The concentration of lysozyme in camel milk (15 μg 100 mL−1) was higher than that in bovine milk (7 μg 100 mL−1). Immunological cross reactions between camel and bovine minor milk proteins were very limited.

The potential of immobilized cell technology to produce freeze-dried, phage-protected cultures of Lactococcus lactis

Abstract Lactococcus lactis cells were immobilized in calcium alginate beads and added to growth media to enable biomass production in the gel. The immobilized population was then freeze-dried. Survival during freeze-drying (FD), stability upon high-temperature storage and residual humidity were evaluated. This culture was compared to a classical liquid starter and fresh beads of immobilized L. lactis in simulated quarg manufacture. Acidifying characteristics, proteolytic activity, sensitivity to bacteriophage and sensory properties of quarg products were evaluated. The population in the beads prior to FD was 7 × 1010 CFU/g. The best survival rate to FD (62–79%) was obtained when the beads were mixed with a milk-based protective solution. In the absence of protective ingredients (milk solids, sucrose and vitamin C) the alginate beads had high water content following FD. Survival of the immobilized cells during FD was as high for immobilized cells as that of free cells. Stability of the immobilized cells at high-temperature storage (45–55°C) was higher than for the free cells. Quarg cheese was succesfully produced in 5 h with the immobilized freeze-dried (IFD) culture, but sensory evaluation confirmed a significant texture difference between cheeses made with free or IFD cultures. Higher inoculation rates were required with the IFD culture to obtain the same acidifying activity as classical fresh liquid starters. The IFD culture performed well under phage contamination; following a 6-h fermentation, 30% of the cells remained viable and active in the phage-contaminated sample. Increase in non-protein amino compounds (0·2 g/100 g cheese) over a 30-day storage period at 4°C was similar in quarg cheeses made with fresh or IFD starters, despite the higher inoculation rate used with the IFD culture.