Nancy J. Gardner

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.

The effect of protective ingredients on the survival of immobilized cells of Streptococcus thermophilus to air and freeze-drying

Streptococcus thermophilus cultures were grown either on trehalose or lactose, immobilized in alginate beads, dipped in various protective solutions and dried by either convection air-drying (CAD) or freeze-drying (FD). Immobilized cultures dipped in the 0.1% peptone solution did not show good survival to CAD or FD, as mortality was over 99%. There was no significant difference in mortality levels, in both methods of drying, when lactose or trehalose were used as protective ingredients. The highest survival levels (50 to 98%) were with a whey-sucrose protective medium, but this was potentially related to a higher pH and solids of the solution. Mortality levels were higher in FD than CAD, and this did not appear to be related to the fact that FD cultures had lower residual moisture contents than those dried under CAD. Cells grown on lactose had slightly higher survival rates to drying than those obtained from CAD. Trehalose-positive and trehalose-negative cultures of S. thermophilus did not show different mortality patterns to CAD or FD.

Fermentation technologies for the production of exopolysaccharide-synthesizing Lactobacillus rhamnosus concentrated cultures

The exopolysaccharide (EPS)-producing cultures such as Lactobacillus rhamnosus RW-9595M present a challenge for the culture producers because the high viscosity of the fermented growth medium makes it difficult to recover the cells by centrifugation or filtration. This study examined four approaches to reduce viscosity of the medium while producing high cell densities: incubation temperature, extended incubation in the stationary growth phase, production in alginate gel beads and fed-batch fermentation technology. Automated spectrophotometry (AS) was used to study the effects of temperature, pH and lactate level on growth of the strain. In AS assays, there was no significant difference in final maximal biomass

Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures.

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l ,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit. Journal of Industrial Microbiology & Biotechnology (2002) 28, 291–296 DOI: 10.1038/sj/jim/7000245

The use of Petrifilm™ for the enumeration of lactococci

Abstract Petrifilm™ aerobic plate counts were compared to Elliker and M17 agar counts for the enumeration of Lactococcus strains. There were no significant differences between counts obtained with the three methods, which suggests that Petrifilm™ plates can be used as a time-saving method for the determination of lactococci starter populations. The standard method of using Petrifilm™, in which 1 ml of sample is analysed, can be modified to have multiple inoculations on the same dry medium culture plates, such that four aliquots of 0·1 ml are added to a single Petrifilm™ plate. Counts obtained with the multiple inoculation method did not differ significantly from the standard single inoculation method recommended by the Petrifilm™ manufacturer.

The spot test method for the in-plant enumeration of bacteriophages with paired cultures of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus

Abstract Using M17 and acidified MRS media for the selective development of Streptococcus salivarius subsp. thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), it was possible to determine phage populations in plates seeded with the paired cultures using the spot test or the standard double layer plaque (DLP) method. There were no significant differences in the enumeration of L. bulgaricus phage 448-c5 on acidified MRS agar when a pure culture of L. bulgaricus 448 or a paired culture of L. bulgaricus 448 and S. thermophilus 1151 were inoculated in the top agar layer. Population readings of S. thermophilus phage 1151-B7 on M17 agar were 0.2 log lower with paired cultures compared to the pure S. thermophilus 1151 strain. Nevertheless, a good correlation (R2 = 0.92) was achieved between phage counts obtained between the two host inoculation procedures. The spot test was compared to the standard DLP method for the enumeration of phages of the two thermophilic cultures. There were no significant differences in the enumeration of L. bulgaricus phage 448-c5 on acidified MRS agar when spot or standard DLP methods were used to determine the phage titre. Population readings of S. thermophilus phage 1151-B7 on M17 agar were slightly lower using the spot test, but correlation between counts obtained between the spot and standard DLP methods was acceptable (R2 = 0.80). Petri plates preinoculated with the host organism could be stored at 4 °C for 18 days and used for a spot test determination of phage titres. The spot test is proposed as a time-saving and easy method for the in-plant enumeration of phages of paired cultures of S. thermophilus and L. bulgaricus.

Population yields and vegetable juice fermentation of Leuconostoc mesenteroides cultures grown under free-cell or immobilized-cell technologies.

Leuconostoc mesenteroides BLAC cultures were produced using classical free-cell fermentation technology and immobilized cell technology (ICT). The ICT process consisted of entrapping the culture into alginate or pectin beads, adding them to a growth medium, and incubating for 14 h to allow growth of the culture inside the beads. Cell populations, survival to freeze-drying and specific acidifying activity (SAA) following inoculation in vegetable juices were examined. In free cell fermentations, 1.3 × 1010 cells/mL were obtained while 1.6 to 2.0 × 1011 cells/g were found in beads. The total cell counts recovered per fermentor were similar in free-cell and ICT systems. In the ICT process, the free-cell level was only of 1% or less. No difference in population yields were noted between alginate and pectin gels. Survival to freeze-drying was between 65 and 80% and was not significantly affected by the prior fermentation process nor the alginate or pectin matrix. A methodology was developed to ascertain the free-cell content of the freeze-dried ICT cultures, and data showed that they contained 97.5% of gel-entrapped cells. Freshly prepared free-cell suspensions had four times higher SAA than comparative free-cell freeze-dried cultures. The dried ICT cultures had lower SAA than free-cell equivalents, but there was no significant effect of the ICT matrix (alginate or pectin) on SAA. The powders of the ICT culture were ground and sieved to obtain particles ranging from 38 μm to 1000 μm in diameter. The smaller the particle, the higher was its SAA. Fermentation of vegetable juices or pastes were carried out with the free and ICT cultures. In the ICT fermentation, the free-cell content of the inoculum represented 2.5% of the total population, but this increased to 88% after the 22 h incubation in vegetable juice. Data are discussed in relationship with potential benefits of ICT cultures in vegetable fermentations.

Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

SummaryImmobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain aw readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g−1 of bead) was followed during storage. Viability losses were high at aw readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 aw solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 aw glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.

Determination of Viable Bacterial Populations in Raw Milk within 20 Minutes by Using a Direct Epifluorescent Filter Technique

The results from a shortened procedure for the direct epifluorescent filter technique (DEFT) determination of viable bacterial populations in raw milk were compared to standard plate counts. Shortening the prefiltration trypsin-Triton X-100 incubation period from 10 to 3 min enabled the completion of the analysis within 20 min. The short DEFT method results had a correlation coefficient (r) of 0.81 with plate counts. With respect to precision, the average difference between values of duplicate plate count analyses was 0.16 log units; that of the short DEFT was 0.14 log units. The slopes of the regressions equations were less than 1, indicating that a direct correlation is not achieved. Short DEFT values were 0.17 log units higher than those of plate counts on milk samples containing less than 10,000 CFU/ml. For milk samples containing counts over 10,000 CFU/ml, short DEFT values averaged only 0.05 log units above plate count readings. Daily preparation of the stain appears unnecessary since acridine orange solutions stored for up to 2 days at 4°C did not produce results significantly (P > 0.05) different from those obtained with fresh solutions. The short DEFT method has potential for the assessment of the bacteriological quality of raw milk in tanker deliveries.