Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC?), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC?) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations—concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate—induced different degrees of transience from VC+ to VC? states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting.     

Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25 degrees C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4 degrees C, followed by treatment in water or chlorine solution at 21 degrees C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers. as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodgedin protective structures on the surface and within tissues of apples.     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

弱后酸化酸奶发酵剂的筛选及应用

选取3种酸奶发酵剂09D-B、YT-A和YT-B,制成酸奶,测定不同温度条件下3种酸奶样品在发酵过程和保质期内的酸度、黏度、pH值和乳酸菌活菌数的变化趋势,研究不同发酵剂对酸奶产品品质的影响。结果表明,发酵剂YT-A的后酸化能量最弱,在4℃冷藏20 d后,酸度仅上升了9°T,产品黏度较高,在冷藏条件下乳酸菌活菌数为4.6×107个/mL,在4℃冷藏和20℃常温贮存过程中,产品的感官状态和组织状态均优于发酵剂09D-B和YT-B。     

Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction

One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.     

Direct microscopic observation of viability of Campylobacter jejuni on chicken skin treated with selected chemical sanitizing agents

The objective of this research was to determine the effect of chlorine, acidified sodium chlorite, and peracetic acid treatments on viable Campylobacter jejuni located at various depths within follicles or folds of chicken skin. Chicken skin was inoculated with C. jejuni transformed with P(c)gfp plasmid (GFP-Campylobacter), which also codes for kanamycin resistance. Effectiveness of sanitizer treatments was determined by plate count. C. jejuni were also observed on chicken skin by confocal scanning laser microscopy, whereby viable and nonviable cells were differentiated by their ability to take up staining with 5-cyano-2,3-ditolyl tetrazolium chloride. Sodium hypochlorite, peracetic acid, and acidified sodium chlorite were each applied at 40 or 100 ppm for 2 or 15 min. Each sanitizer resulted in approximately a 1-log decrease (CFU) when used at 100 ppm for 15 min and no significant decrease when used at 40 ppm for 2 min. Numbers of viable cells observed on the skin by direct microscopic count were similar to numbers obtained by plate count. Although viable counts decreased with sanitizer treatments, the total number of Campylobacter cells (live plus dead) attached to the skin remained unchanged. After each chemical treatment, viable C. jejuni were observed at depths of 0 to 10, 11 to 20, and 21 to 30 microm in folds or follicles of chicken skin. Most of the C. jejuni that survived treatment were located at 0 to 10 microm depth, which is where most of the viable cells were located before treatment. The inability of chemical sanitizers to effectively eliminate C. jejuni on chicken skin does not appear to be a result of protection by location in feather follicles or other depressions in the skin.     

In vitro evaluation of Lactobacillus gasseri strains of infant origin on adhesion and aggregation of specific pathogens

Numerous Lactobacillus species are members of the normal healthy human intestinal microbiota, and members of the Lactobacillus family predominate among the current marketed probiotic strains. Most of the current commercial probiotic strains have not been selected for specific applications but rather have been chosen based on their technological properties. Often the ability of such strains to temporarily colonize the gastrointestinal tract may be lacking, and the interactions with intestinal microbiota are few. Furthermore, the competitive exclusion properties of potential probiotic bacteria are strain specific and vary greatly. Thus, it is highly desirable that new candidate probiotic isolates originate from the healthy target population. In this study, seven newly isolated strains of Lactobacillus gasseri originating from feces of a healthy newborn child were evaluated for their ability to adhere to intestinal mucus, to autoaggregate and coaggregate with the model pathogens Cronobacter sakazakii (ATCC 29544) and Clostridium difficile (1296). All the bacterial strains, single or in combination, in viable and nonviable forms, were able to autoaggregate. The coaggregation with C. sakazakii or C. difficile was higher (P < 0.05) in nonviable than in the viable forms. Single L. gasseri strains showed similar adhesion abilities to intestinal colon mucus. The seven L. gasseri strains when combined were also able to significantly compete with, displace, and inhibit the adhesion of C. sakazakii and C. difficile in the mucus model. This study demonstrates that the studied L. gasseri strains fulfill the basic adhesion and aggregation properties for probiotics and could be considered for potential future use in children.     

Effect of lactobacillus adjunct cultures on the microbiological and physicochemical characteristics of Roncal-type ewes'-milk cheese

The effect of two different experimental adjunct cultures composed of native facultatively heterofermentative lactobacilli (FHL) on the development of various groups of micro-organisms in Roncal-type ewes' milk cheese was studied. Four cheese batches were manufactured from raw milk (C), pasteurized milk (P), pasteurized milk and an adjunct culture of Lactobacillus paracasei (PP); and pasteurized milk and adjunct culture of Lactobacillus paracasei plus Lactobacillus plantarum (PPP). Retention of the two adjunct cultures in the cheeses was good, and population levels remained constant at around 10(7) cfu g(-1) of cheese throughout ripening. Levels of Enterobacteriaceae and enterococci fell off more abruptly in the batches made with the Lactobacillus adjunct cultures, suggesting competition between the added lactobacilli and those groups of micro-organisms. The inhibitory effect was greater for the adjunct culture composed of L. paracasei plus L. plantarum. Lactococcal levels were higher in the batches made with added FHL, which may be indicative of a synergistic effect between these two groups.     

Comparative evaluation of yogurt and low-fat cheddar cheese as delivery media for probiotic Lactobacillus casei

This study used Lactobacillus casei 334e, an erythromycin-resistant derivative of ATCC 334, as a model to evaluate viability and acid resistance of probiotic L. casei in low-fat Cheddar cheese and yogurt. Cheese and yogurt were made by standard methods and the probiotic L. casei adjunct was added at approximately 10(7) CFU/g with the starter cultures. Low-fat cheese and yogurt samples were stored at 8 and 2 degrees C, respectively, and numbers of the L. casei adjunct were periodically determined by plating on MRS agar that contained 5 microg/mL of erythromycin. L. casei 334e counts in cheese and yogurt remained at 10(7) CFU/g over 3 mo and 3 wk, respectively, indicating good survival in both products. Acid challenge studies in 8.7 mM phosphoric acid (pH 2) at 37 degrees C showed numbers of L. casei 334e in yogurt dropped from 10(7) CFU/g to less than 10(1) CFU/g after 30 min, while counts in cheese samples dropped from 10(7) CFU/g to about 10(5) after 30 min, and remained near 10(4) CFU/g after 120 min. As a whole, these data showed that low-fat Cheddar cheese is a viable delivery food for probiotic L. casei because it allowed for good survival during storage and helped protect cells against the very low pH that will be encountered during stomach transit.     

COMPARISON OF METHODS FOR THE DETERMINATION OF CELL VIABILITY IN STORED BAKER'S YEAST

The classical plate method for discriminating between viable and nonviable yeast cells in stored baker's yeast was compared with dead cell staining techniques using methylene blue and three fluorochrome stains. The increase of dead yeast cells during storage of baker's yeast for up to 16 days at 5°C, 20°C and 35°C was determined. During prolonged storage, especially at 35°C, the death rate increased rapidly and the yeast cake began to soften because of autolysis. In these conditions the choice of method for determining the proportion of dead cells proved to be of great importance. Useful results for yeast stored for some time at 35°C could be obtained only by the fluorochrome technique using primuline, acridine orange or acriflavine as fluorochromes.     

Influence of adjuncts as a debittering aids in encountering the bitterness developed in cheese slurry during accelerated ripening

An attempt was made to accelerate the flavour development in cheese base with the help of exogenous proteolytic and lipolytic enzymes (1:1 proportion, each at the rate of 0.025% by weight of cheese‐base) and ripening at elevated temperatures (i.e. 20 ± 1 °C) for up to 12 days. To counter the bitterness developed, adjunct cultures were used: viable or attenuated (freeze‐shocked or heat shocked). Study of biochemical characteristics, electrophoretic pattern and sensory evaluation of the product were carried out. An acceptable enzyme‐modified, lightly salted cheese base was obtained using 0.025% each of proteolytic and lipolytic enzymes, along with 5% starter culture and adjuncts followed by ripening up to 12 days. Freeze‐shocked adjunct Lactobacillus helveticus produced enzyme‐modified cheese base with no detectable bitterness. The usage of exogenous enzymes, temperature of ripening, ripening period and interactions amongst these parameters had significant (P < 0.01) influence on all of the biochemical characteristics monitored.     

Spray‐drying for the production of dried cultures

This review emphasises the importance of spray‐drying as an under‐used but promising technique to preserve viable and active starter cultures and also, potentially, probiotic cultures. The knowledge concerning the production of spray‐dried starter cultures is discussed. Different drying techniques and micro‐organisms have been investigated for their survival through the drying process. During drying and subsequent storage in the dried state, bacteria are subjected to several stresses, which have already been described as causing damage to cells, leading to the loss of cellular viability and activity. Some studies found that several factors/strategies can confer improved cellular viability.     

Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce

Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

Rapid identification and quantitation of the viable cells of Lactobacillus casei in fermented dairy products using an aptamer-based strategy powered by a novel cell-SELEX protocol

An aptamer-based strategy was developed for qualitative and quantitative analysis of viable Lactobacillus casei in dairy products. Three highly specific aptamers for L. casei were obtained using systematic evolution of ligands by exponential enrichment protocol using the whole bacterium cell as the target (cell-SELEX) facilitated by polyethyleneglycol and chitosan modified graphene oxide and complementary ring-mediated rolling circle amplification. Two aptamers, one for separating and enriching the L. casei cells and the other for generating fluorescence signals, were employed to develop an aptamer-based strategy, which was demonstrated for the selective detection of L. casei in commercial dairy drinks, with a dynamic range of 10⁵ to 10⁹ cfu/mL. Viable and nonviable L. casei cells could be discriminated based on the significant difference in fluorescence intensity. This established strategy is of high selectivity and sensitivity, and can be used for rapid analysis of viable L. casei in quality control and food surveillance areas.     

Reduction of patulin in aqueous solution by lactic acid bacteria

This study aims to investigate the ability of lactic acid bacteria (LAB) to remove patulin (PAT) from aqueous solution with respect to the bacterial viability, initial PAT concentration, incubation time, temperature, and pH. The removal of PAT determined by high-performance liquid chromatography (HPLC) coupled with UV detector. The maximum PAT uptake was achieved by Bifidobacterium bifidum 6071 and Lactobacillus rhamnosus 6149 strains (52.9% and 51.1%) for viable and (54.1% and 52.0%) for nonviable cells after 24 h incubation. The highest removal of PAT was at pH 4.0 and 37 °C and increased with decreasing of toxin levels. The removal ability of selected strains could represent new strategies for a possible application in contaminated food products and animal feed.     

Potential of anticlostridial Lactobacillus isolated from cheese to prevent blowing defects in semihard cheese

Five anticlostridial Lactobacillus strains isolated from cheese were selected for a mixed adjunct culture. Cheese with the mixed adjunct culture (experimental) and without (control) was made in triplicate and ripened as vacuum‐packed and surface‐ripened cheese. Cheese gross composition was similar. Excessive gas formation occurred only in control cheeses. In contrast to control cheeses, the experimental cheeses were dominated by the added adjunct Lactobacillus strains (repetitive‐PCR). Casein breakdown was not influenced, however, the total amount of amino acids and pH was slightly lower in the experimental cheeses. Anticlostridial nonstarter Lactobacillus strains have potential as protective adjunct cultures against blowing defects in cheese.     

Influence of adjunct cultures on volatile free fatty acids in reduced-fat Edam cheeses

The effects of the adjunct cultures Lactococcus lactis ssp. diacetylactis, Brevibacterium linens BL2, Lactobacillus helveticus LH212, and Lactobacillus reuteri ATCC 23272 on volatile free fatty acid production in reduced-fat Edam cheese were studied. Lipase activity evaluation using p-nitrophenyl fatty acid ester substrates indicated that L. lactis ssp. diacetylactis showed the highest activity among the 4 adjunct cultures. Full-fat and 33% reduced-fat control cheeses (no adjunct) were made along with 5 treatments of reduced-fat cheeses, which included individual, and a mixture of the adjunct cultures. Volatile free fatty acids of cheeses were analyzed using static headspace analysis with 4-bromofluorobenzene as an internal standard. Changes in volatile free fatty acid concentrations were found in headspace gas of cheeses after 3-and 6-mo ripening. Acetic acid was the most abundant acid detected throughout ripening. Full-fat cheese had the highest relative amount of propionic acid among the cheeses. Certain adjunct cultures had a definite role in lipolysis at particular times. Reduced-fat cheese with L. lactis ssp. diacetylactis at 3-mo showed the highest levels of butyric, isovaleric, n-valeric, iso-caproic, and n-caproic acid. Reduced-fat cheese with Lactobacillus reuteri at 6 mo produced the highest relative concentration of isocaproic, n-caproic, and heptanoic, and the highest relative concentration of total acids.     

Evaluation of changes during storage of probiotic dahi at 7°C

Low-fat probiotic dahi (an Indian traditional fermented milk product like yogurt) prepared with lactococci starter and two adjunct probiotic cultures of Lactobacillus acidophilus and Lactobacillus casei was stored at 7°C. The survival of bacterial species was affected during storage of dahi, in which viable counts increased up to 2 days and gradually decreased from 2 days, up to 8 days. The quality of refrigerated dahi was assessed by a panel of trained judges. On storage, pH significantly decreased over storage time, indicating that the dahi samples did not develop much acidity under the storage conditions of the study. However, after 8 days of storage, the samples were disliked by the panel, which reported slight bitterness, which may have been due to proteolysis. Proteolysis increased during storage. More α_s1-casein was degraded than β-casein. On the basis of the results of this study we can conclude that dahi stored up to 8 days may be acceptable to consumers.