Technological and functional Technological and functional studies of candidate

VERA PINGITORE E.; JUÁREZ TOMÁS M. S.; SESMA F.; NADER-MACIAS M.E.

Sevilla - España

Congreso; BioMicroWorld; 2007

The infections of the female urogenital tract constitute a serious social problem and of public health, because their high prevalence (around 1 billion of persons per year in the world) and the high health-care costs involved. These infections cause high morbi-mortality rates in women and new born children, mainly in countries where the AIDS (acquired immunodeficiency syndrome) is a prevalent disease. The use of probiotic products to prevent urogenital infections can be an effective alternative to the conventional antibiotic therapy. Probiotic microorganisms, such as lactobacilli, can be able to restore the balance of urogenital microflora. In order to select probiotic microorganisms to be included in vaginal probiotic products, human vaginal Lactobacillus strains were isolated and identified in previous works. These microorganisms were characterized in terms of potentially probiotic properties and growth performances. The main objective of our research group is the design of a probiotic pharmaceutical product containing different Lactobacillus strains combined with salivaricin, a bacteriocin produced by Lactobacillus salivarius CRL 1328. For the pharmaceutical industry, the technological properties of the strains are of main interest, because a form suitable for product applications and long stability is highly desirable, settled on the large-scale production of concentrated cultures and bacteriocin. Then, a properly lyophilized formulation must maintain adequate number of viable bacteria and physical and chemical stable proteins during shipping and long-term storage, even at  room temperatures. The aims of this work were to study the effects of different protective agents on: a) the expression of beneficial characteristics of six freeze-dried vaginal lactobacilli, during two years of storage; b) the stability of salivaricin during lyophilization and storage at different temperatures. For the first objective, Lactobacillus cells were freeze-dried in the following protectors: 12% (w/v) sucrose, 12% lactose, 6% reconstituted skim milk (RSM), and 6% RSM added with 12% sucrose or 12% lactose, and stored at 4°C. At different storage times, lyophilized cultures were rehydrated and grown in LAPTg broth during 24 h at 37°C before testing each probiotic property (auto-aggregation capability; production of lactic acid, hydrogen peroxide, and bacteriocin). For the recovery of salivaricin activity after freeze-drying, the following substances were examined: 5% sucrose, 4% sodium glutamate, 4% mannitol + 1% sucrose, 5% RSM, 5% sucrose + 0.5% PEG (polyethilenglycol 8000), 0.4% Tween 80, 2% ascorbic acid. To determine the protective efficiency during the subsequent storage of bacteriocin in dried state, samples were kept at 25ºC, 4ºC and –20ºC for eight months. In all the samples the proteins concentration, inhibitory activity and weight were determined. The results shows that the six vaginal lactobacilli evaluated maintain their capabilities of antimicrobial substances production or auto-aggregation, after the freeze-drying process and during the storage for 2 years. In the case of bacteriocin, lactic acid or H2O2 producers, the variation of the levels of inhibitory substances was dependent on the Lactobacillus strains, protective media and storage time. However, the extent of auto-aggregation of L. johnsonii CRL 1294 was high in all the conditions assayed and storage times. On the other hand, the stability of freeze-dried bacteriocin was affected by the storage temperature and the protectors employed. The higher recuperation of specific activity, expressed as Arbitrary Units (AU)/ug proteins or AU/mg powder, was observed at 4°C and -20°C. At the different temperatures assayed, the higher protection was observed in presence of PEG + sucrose and ascorbic acid. At 4°C and -20°C, the recovery of bacteriocin activity during storage in presence of mannitol, sucrose or glutamate was not significantly different compared to control. RSM shown to be ineffective in protecting the bacteriocin during storage at the three temperatures evaluated. In conclusion, certain additives were more effective that others in promoting the conservation of functional characteristics of vaginal lactobacilli or bacteriocin stability after freeze-drying. These results are of main importance for the future design of pharmaceutical formulations to prevent urogenital infections. Keywords vaginal probiotic; freeze-drying; protectants; functional activities; bacteriocin stability