Andrea Quiberoni

Bacteriophages and dairy fermentations.

This review highlights the main strategies available to control phage infection during large-scale milk fermentation by lactic acid bacteria. The topics that are emphasized include the factors influencing bacterial activities, the sources of phage contamination, the methods available to detect and quantify phages, as well as practical solutions to limit phage dispersion through an adapted factory design, the control of air flow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures.

Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides

The effect of several biocides and thermal treatments on the viability of four Lactobacillus delbrueckii phages was investigated. Time to achieve 99% inactivation of phages at 63 and 72 degrees C in three suspension media (Tris Magnesium Gelatin (TMG) buffer, Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)) was calculated. Thermal resistance depended on the phage considered, but a marked heat-resistance was exhibited by one phage (Ib(3)) since its high titre suspensions were completely inactivated only after 45 min at 72 degrees C or 15 min at 90 degrees C. A clear protective effect of the milk was revealed when the three suspension media were compared. As regards to the effects of biocides on phages, only peracetic acid was found to be effective for inactivating high titre suspensions. Ethanol, even at a concentration of 100%, was not suitable to assure no surviving phage particles and isopropanol turned out to be less effective than ethanol. Sodium hypochlorite at 200-400 ppm inactivated the phages completely, except phage Ib(3), which was only destroyed after treatments with 1200 ppm. The diversity observed in the heat and biocide resistance of L. delbrueckii phages is useful to establish a basis for adopting the most effective thermal and chemical treatments for inactivating them in dairy plants and laboratory environments.

Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains

Aim:  To investigate the influence of several environmental factors on the viability and cell‐adsorption for two Lactobacillus casei/paracasei bacteriophages (PL‐1 and J‐1).

Inactivation of Lactobacillus helveticus bacteriophages by thermal and chemical treatments.

The effect of several biocides and thermal treatments on the viability of four Lactobacillus helveticus phages was investigated. Times to achieve 99% inactivation of phages at 63 degrees C and 72 degrees C in three suspension media were calculated. The three suspension media were tris magnesium gelatin buffer (10 mM Tris-HCl, 10 mM MgSO4, and 0.1% wt/vol gelatin), reconstituted skim milk sterile reconstituted commercial nonfat dry skim milk, and Man Rogosa Sharpe broth. The thermal resistance depended on the phage considered, but a treatment of 5 min at 90 degrees C produced a total inactivation of high titer suspensions of all phages studied. The results obtained for the three tested media did not allow us to establish a clear difference among them, since some phages were more heat resistant in Man Rogosa Sharpe broth and others in tris magnesium gelatin buffer. From the investigation on biocides, we established that sodium hypochlorite at a concentration of 100 ppm was very effective in inactivating phages. The suitability of ethanol 75%, commonly used to disinfect utensils and laboratory equipment, was confirmed. Isopropanol turned out to be, in general, less effective than ethanol at the assayed concentrations. In contrast, peracetic acid (0.15%) was found to be an effective biocide for the complete inactivation of all phages studied after 5 min of exposure. The results allowed us to establish a basis for adopting the most effective thermal and chemical treatments for inactivating phages in dairy plant and laboratory environments.

The lactic acid microflora of natural whey starters used in Argentina for hard cheese production

Abstract A total of 56 samples of natural whey starters, obtained by two sampling plans and employed for Argentinian hard cheese production, were examined. Seasonal changes in technological (pH, acidity, and acidifying and proteolytic activities) and microbiological (lactic acid microflora characteristics) parameters were analyzed. Furthermore, phage resistance and IS 1201 identification were studied among Lactobacillus helveticus isolates. Although technological characteristics of the cultures did not show significant variations, the relative proportion of Lb. helveticus and Lb. delbrueckii subsp. lactis was different between sampling 1 and 2. As a consequence, frequences of fast, intermediate and slow bacterial variants were also variable. On the other hand, lactic acid microflora showed a rather low tolerance to NaCl. Among Lb. helveticus strains, phage resistance was widespread and the presence of IS 1201, a specific insertion sequence for this species, was also demonstrated.

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption

Aims:  Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized.

Thermophilic Lactic Acid Bacteria Phages Isolated from Argentinian Dairy Industries

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradleys group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

Reggianito Argentino cheese: influence of Lactobacillus helveticus strains isolated from natural whey cultures on cheese making and ripening processes

Abstract Reggianito Argentino cheeses were manufactured with three defined single strains Lactobacillus helveticus cultured in sterile whey, and one “natural” whey starter. Gross composition of cheeses did not significantly differ, and viable starter cell counts were similar for all cheeses. Soluble nitrogen at pH 4.6 was also alike for cheeses made with natural or selected starters, but soluble nitrogen in trichloroacetic acid 12% and phosphotungstic acid 2.5% showed significant differences. Electrophoretograms showed that γ casein bands increased during ripening, while β casein band decreased; α s1 casein was also cleaved to α s1 -I. Acid degree values of fat for control and experimental 0-day old cheeses were slightly different, but were similar at 90 and 180 days of ripening. All cheeses were good quality Reggianito, but control and experimental samples differed in aroma and texture. Natural whey starter replacement by selected single starter of Lactobacillus did not alter cheese making and primary proteolysis or lipolysis, but it modified secondary proteolysis and sensory characteristics.

Review: Efficiency of Physical and Chemical Treatments on the Inactivation of Dairy Bacteriophages

Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO2 has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions.

Genetic (RAPD‐PCR) and technological diversities among wild Lactobacillus helveticus strains

Diversity in 25 Lactobacillus helveticus strains isolated from natural whey cultures for Argentinian hard cheese production was studied by means of RAPD‐PCR patterns and technological parameters (acidifying and proteolytic activities, salt tolerance, diacetyl, H2O2 and slime production, phage sensitivity). In the RAPD diversity study, 10 Lact. helveticus strains from the CNRZ collection were also included.