Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.     

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.     

Influence of nonfermented dairy products containing bacterial starter cultures on lactose maldigestion in humans

The effect of nonfermented dairy products containing yogurt or acidophilus cultures on lactose utilization by lactose-maldigesting humans was investigated. Yogurt and acidophilus milk containing 10(7) or 10(8) of Streptococcus thermophilus and Lactobacillus bulgaricus, or Lactobacillus acidophilus, respectively, were prepared using commercially processed 2% low fat milk. Immediately following inoculation, products were refrigerated. Lactose maldigestion was monitored by measuring breath hydrogen excretion at hourly intervals for 8 h following consumption of 400 ml of each test meal containing approximately 20 g of lactose. The yogurt milk containing 10(8) cfu/ml was shown to contain significant concentrations of microbial beta-galactosidase (EC 3.2.1.23; approximately 3 U/ml), which remained stable for at least 14 d at refrigerator temperatures. Breath hydrogen peaks were delayed and significantly lower (approximately 20 ppm at 5 to 7 h) than control values (approximately 70 ppm at 4 h), and intolerance symptoms were eliminated in all subjects. Yogurt milk containing 10(7) cfu/ml demonstrated intermediate breath hydrogen values and was marginally significantly different from control values. Lactobacillus acidophilus strains with varying resistance to bile and total beta-galactosidase-producing potential were also tested. Only one strain, LA-1, which demonstrated low bile resistance and intermediate beta-galactosidase activity, was capable of significantly decreasing breath hydrogen values when 10(8) cfu/ml of milk was consumed.     

Characterization and selection of Lactobacillus sakei strains isolated from traditional dry sausage for their potential use as starter cultures

A total of 36 Lactobacillus sakei strains isolated from traditional dry sausage, produced without the use of starter cultures, were characterized in respect to their technological properties, i.e. growth and acidification kinetics, ability to grow at different temperatures, pH values or salt concentrations, gas production from glucose, heme-dependent catalase activity, inhibitory activity against pathogens and proteolytic and lipolytic activities.Growth rates were comprised between 0.12 and 0.55 gen/h; while acidification rates varied from 0.01 and 0.07 pH unit/h. All isolates were able to grow at pH 4.2–9.6 and at 15 °C on acetate agar supplemented with 4% NaCl, but were not able to grow at 0 and 45 °C and with 10% NaCl in the same pH conditions. However, 97% of the isolates were able to grow at 4 °C, but only 11%, and 55% were able to grow at pH 3.9 and with 6.5% NaCl, respectively. All isolates were homofermentative from glucose. Heme-dependant catalase activity was found for 97% of the isolates and 3% displayed antibacterial activity against Listeria innocua. Most isolates showed leucine and valine arylamidase and only one exhibited a lipase (C14) activity.Two strains were selected using a multi-variate analysis. Both of them showed a panel of properties highly desirable in sausage fermentation. We propose the use of these strains, in combination with coagulase-negative staphylococci as starter cultures in the studied small-scale facility in order to improve and standardize sausage safety and quality while preserving their typicality.     

Evaluation of the possible use of potentially probiotic Lactobacillus strains in dairy products

In the study, the ability of two potentially probiotic strains Lactobacillus plantarum 14 and Lactobacillus fermentum 4a to milk fermentation and the possibility to use them in yogurt production were investigated. The strains did not acidify milk during 24 h and 72 h fermentation at 37C, but grew well and remained at the level of 10⁸ colony-forming units (CFU)/mL during 21 days of cold storage. Their application to yogurt production along with commercial starter culture consisted on L. delbrueckii ssp. bulgaricus and S. thermophilus allowed to obtain products with typical sensory properties, pH values and numbers of potentially probiotic bacteria at desired level 10⁷ CFU/mL.     

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products

Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products. For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp. (eight of each) were used. The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics. A variety of interactions was demonstrated. Lb. delbrueckii subsp. bulgaricus was found to be able to inhibit S. thermophilus strains. Among probiotic cultures, Lb. acidophilus was the sole species that was inhibited by the others (Lb. casei and Bifidobacterium). In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions. The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them). The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage.     

Mezcal: indigenous Saccharomyces cerevisiae strains and their potential as starter cultures

Food Science and Biotechnology - In this study, 19 indigenous mezcal Saccharomyces cerevisiae strains were screened for their tolerance to grow under different stress conditions and their potential...     

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.     

Screening of commercial meat starter cultures at low pH and in the presence of bile salts for potential probiotic use

The survival of lactic acid bacterial strains from eight meat starter cultures under conditions similar to those in the gastrointestinal tract was determined. The conditions in stomach were simulated by using pH 1-5 phosphate-buffered saline. The conditions in small intestine were simulated by using MRS broth over a pH range 4-7 and two bile salt concentrations (0.15% and 0.30%). The survival capacity at pH 3 was strain dependent. 0.30% bile salts were critical for screening bile salt tolerant strains at pH 6. Strains of Lactobacillus sake (RM10) and Pediococcus acidilactici (P2) had the best survival capacities under acidic conditions and at the higher concentration of bile salts.     

Isolation and characterization of Lactobacillus species having potential for use as probiotic cultures for dogs

ABSTRACT:  The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus . There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not.     

发酵香肠益生型乳酸菌发酵剂的筛选

从主要自然发酵肉制品中分离、纯化得到77株耐酸耐胆汁的乳酸菌,并进一步对分离菌株进行了耐高酸(pH2.5)、0.3%胆盐的生长实验.25株在pH值2.5,培养3 h后表现出较高存活性,21株经低酸预处理后在0.3%胆盐存在的情况下生长良好.最后运用API 50CHL对筛选菌株进行了鉴定.这些菌株可作为进一步筛选发酵香肠优良乳酸菌种的出发菌株,以便筛选品质优良的益生性乳酸菌,作为新型发酵香肠发酵剂.     

Screening of dairy yeast strains for probiotic applications

To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.     

Probiotic potential of Lactobacillus strains isolated from dairy products

Twenty-nine Lactobacillus strains of dairy origin were examined in vitro for their probiotic potential. Only a few strains were able to survive at pH 1 or in the presence of pepsin, while all were unaffected by pH 3, pancreatin and bile salts. Strains exhibited variable bile salt hydrolase activity. None was haemolytic. The majority of strains were resistant to vancomycin and teicoplanin, but sensitive to chloramphenicol and tetracycline. A few strains were able to adhere to Caco-2 cells. Although no bacteriocin activity was detected in vitro, strains L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were able to inhibit the adhesion of Escherichia coli and Salmonella typhimurium to Caco-2 cells. They also induced the secretion of pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells. These three strains were therefore found, in vitro, to possess desirable probiotic properties.     

The survival and persistence in the human gastrointestinal tract of five potential probiotic lactobacilli consumed as freeze-dried cultures or as probiotic sausage

Among five lactobacilli (L. plantarum MF1291, MF1298, DC13, L. pentosus MF1300 and L. salivarius DC5) which were administrated as freeze-dried cultures for 17 volunteers, MF1298 and DC13 were the most frequently reisolated strains in faeces demonstrating the human gastric survival of these strains. Furthermore, MF1298 and DC13 persisted in the same volunteer after ended intake, suggesting host-specific persistence behaviour. When MF1298 was administrated as sausage fermented with this strain, the number of volunteers harbouring MF1298 increased from 4 to 10 indicating that the sausage matrix protects the survival through the gastrointestinal tract (GIT).     

Characterization of homofermentative lactobacilli isolated from kefir grains: potential use as probiotic

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S-23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2.5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.     

传统乳制品中潜在益生植物乳杆菌的体外筛选

对从内蒙古牧区乳制品中分离到的29株植物乳杆菌的耐酸性（pH=3．0）、耐胆汁性（质量浓度3g／L牛胆酸钠）、抗菌活性进行了系统性研究。结果表明，有10株菌具有较好的耐酸耐胆盐特性，结合抗菌特性及黏附性试验结果，菌株WZ47-1，WZ32-2-1和WH13-2有可能成为潜在益生菌菌株。