Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.     

Protein Degradation by Lactobacillus plantarum and Lactobacillus casei in a Sausage Model System

ABSTRACT: :
The proteolytic activity of a starter culture involving Lactobacillus plantarum and Lactobacillus casei towards meat sarcoplasmic and myofibrillar proteins during the fermentation of a sausage-like system was studied. After 96 h of incubation the proteolytic system of L. plantarum CRL681 caused a degradation of both sarcoplasmic and myofibrillar proteins, whereas L. casei CRL705 showed a different affinity to meat proteins. The inoculation of both strains showed a higher activity toward sarcoplasmic fraction. These results correlated with a high rate of sarcoplasmic protein degradation observed in SDS-PAGE analysis. The generation of free amino acids as well as the pH drop at the end of the incubation period was maximal in presence of the mixed starter culture, thereby demonstrating the suitability of these strains to be used in the fermentation of meat products.     

干酪乳杆菌基因组改组菌株耐酸性表征

本实验对干酪乳杆菌基因组改组菌株的耐酸性进行了研究,结果表明,冷冻( － 80℃)和临界pH 值(pH3.2)对干酪乳杆菌基因组改组菌株影响不显著,HCl 和游离乳酸对该菌株生长有一定影响,游离乳酸影响更大。在pH5.0 条件下发酵60h,干酪乳杆菌改组菌株获得的菌体干重为6.34g/L,乳酸产量为87.6g/L,比原始菌株耐酸性有显著提高。     

产L-乳酸之乳杆菌的筛选

本研究通过平板划线法及乳酸发酵实验,从干酪及酸菜样品中分离筛选出二株具有较强产L-乳酸能力的乳杆菌。经糖发酵、生长温度、精氨酸发酵产氨、产硫化氢试验及细胞壁中二氨基庚二酸(DAP)成分分析,确认为干酪乳杆菌干酪亚种和干酪乳杆菌鼠李糖亚种。它们在葡萄糖85g/L,乳糖35g/L,酵母膏5g/L,蛋白胨10g/L,NaCl1.3g/L,K2HPO41.5g/L,MgSO4·7H2O0.33g/L,MnSO40.05g/L的培养基中,于37℃下培养48h,产L-乳酸的量分别达到56.50g/L和50.80g/L。     

Dry sausage fermented by Lactobacillus rhamnosus strains

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.     

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.     

Temperature effect on the biological activity of Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251 in pure and mixed cultures grown in soymilk

The influence of temperature on the growth and biological activity of two probiotic strains (Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251) as pure and mixed cultures in soymilk (SM) were evaluated. Maximum growth was observed at 37°C in both mixed and pure cultures. In a product prepared with the mixed culture (1:1) at 37°C, the amount of lactic acid produced was approximately 55 mmol l⁻¹ after 24 h with a slow production rate (2.8 mmol l⁻¹ h⁻¹); the formation of acetic acid was higher with respect to pure cultures (82.01 mmol l⁻¹ after 24 h), and final pH (24 h) was 5.0. About 85% of the total amount of sugars in SM was reduced, mainly sucrose. Stachyose was reduced (71%) after 4 h of incubation. Maximum activity of alpha-galactosidase (alpha-gal) (13.2 U ml⁻¹) was observed after 6 h. At 37°C the bifidobacterium strain was viable in mixed culture throughout the period assayed. At lower (30°C) or higher (42°C) temperatures, mixed culture showed slower growth and lower acid production in SM but the alpha-gal activity was stimulated at 30°C.     

Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese

We evaluated the suitability of Argentinian Fresco cheese as a food carrier of probiotic cultures. We used cultures of Bifidobacterium bifidum (two strains), Bifidobacterium longum (two strains), Bifidobacterium sp. (one strain), Lactobacillus acidophilus (two strains), and Lactobacillus casei (two strains) in different combinations, as probiotic adjuncts. Probiotic, lactic starter (Lactococcus lactis and Streptococcus thermophilus), and contaminant (coliforms, yeasts, and molds) organisms were counted at 0, 30, and 60 d of refrigerated storage. Furthermore, the acid resistance of probiotic and starter bacteria was determined from hydrochloric solutions (pH 2 and 3) of Fresco cheese. The results showed that nine different combinations of bifidobacteria and L. acidophilus had a satisfactory viability (count decreases in 60 d <1 log order) in the cheese. Both combinations of bifidobacteria and L. casei cultures assayed also showed a satisfactory survival (counts decreased <1 log order for bifidobacteria but no decrease was detected for L. casei). On the other hand, the three combinations of bifidobacteria, L. acidophilus, and L. casei tested adapted well to the Fresco cheese environment. When a cheese homogenate at pH 3 was used to partially simulate the acidic conditions in the stomach, the probiotic cultures had an excellent ability to remain viable up to 3 h. At pH 2, the cell viability was more affected; B. bifidum was the most resistant organism. This study showed that the Argentinian Fresco cheese could be used as an adequate carrier of probiotic bacteria.     

Antifungal activity of sodium acetate and Lactobacillus rhamnosus

The inhibition of molds by sodium acetate in deMan Rogosa Sharpe (MRS) medium, along with the antifungal activity of Lactobacillus rhamnosus VT1, was studied by the slope agar plate method. MRS agar prepared with and without sodium acetate was used as the agar substrate. A total of 42 strains of Aspergillus, Penicillium, Fusarium, Alternaria, Cladosporium, and Rhizopus were used to compare sensitivities to the inhibitory activity of sodium acetate and L. rhamnosus VT1. It was found that sodium acetate in MRS medium affected the growth of 33 of the 42 mold strains tested to various degrees. The highest sensitivity to sodium acetate was shown by strains of Fusarium, followed by strains of Penicillium, Aspergillus, and Rhizopus. L. rhamnosus VT1 also inhibited mold growth. A significant finding was that sodium acetate and L. rhamnosus VT1 in combination exhibited a possible synergistic action. Thirty-nine of the 42 mold strains tested were completely inhibited by the presence of both antifungal agents. This finding confirms that sodium acetate, a basic component of commercial MRS medium, has strong antifungal properties, and this must be taken into consideration when evaluating the antifungal activity of Lactobacillus cultures grown in MRS broth.     

Lactobacillus reuteri CRL 1100 as starter culture for wheat dough fermentation

The effect of sucrose on the fermentation balance of Lactobacillus reuteri CRL 1100 and the invertase activity of this strain in wheat dough and culture medium (MRSs) was evaluated. The enzyme activity was dependent on the environmental pH releasing glucose and fructose from sucrose hydrolysis. Glucose was used as carbon source, while fructose was mainly used as electron acceptor to produce mannitol up to 10h of fermentation. Thereafter, fructose seemed to be metabolized by the heterofermentative pathway, which determined an increase in the concentration of acetate (6 mmol l(-1)), lactate (2 mmol l(-1)) and ethanol (1 mmol l(-1)) and the lack of mannitol formation after glucose depletion. The fermentation balance of Lb. reuteri CRL 1100 during the dough fermentation resulted in lower (63%) ethanol, higher (75%) acetate production and soluble carbohydrates concentrations, like MRSs cultures. This fermentation profile would be important to obtain an optimal growth of yeast and the optimal bread flavor and taste.     

Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.     

Mode of action of lactocin 705, a two-component bacteriocin from Lactobacillus casei CRL705

Lactocin 705 is a bacteriocin whose activity depends on the complementary action of two peptides (705alpha and 705beta) of 33-amino-acid residues each and is produced by Lactobacillus casei CRL705. Biologically active, synthetic lactocin 705 was used to study the mode of action on sensitive cells of Lactobacillus plantarum CRL691. The addition of 90 nmol l(-1) of lactocin 705 to cells of L. plantarum dissipated both, the membrane potential (DeltaPsi) and the pH gradient (DeltapH). Energized membrane, obtained after the addition of glucose, were more susceptible to lactocin 705 action leading to the immediate release of intracellular K(+) and inorganic phosphate. When the role of various ions on sensitive cells were analyzed, only Ca(2+) ion exhibited a protective effect against lactocin 705. These data suggest that the presence of a proton motive force (PMF) promotes the interaction of the bacteriocin with the cytoplasmic membrane of energized cells, leading to pore formation which allows for the efflux of ions, thereby ensuring efficient killing of target bacteria.     

白酒窖泥中乳酸菌分离鉴定及其发酵产挥发性风味物质比较

从白酒窖泥中分离得到11株乳酸菌,经16S rDNA基因序列同源性分析,鉴定为1株短乳杆菌、1株鼠李糖乳杆菌、2株干酪乳杆菌和7株铅黄肠球菌。分析了11株菌MRS培养基发酵产乳酸性能,结果表明:乳酸产量与菌群生长呈正相关,鼠李糖乳杆菌L9、干酪乳杆菌L10与L11发酵液乳酸含量较高,分别为15.74、15.58、14.74 g/L。4株代表菌相同条件下发酵高粱培养液产挥发性风味组分,共有物质13种,包含了白酒中重要香气成分:乙酸、己酸、乙酸苯乙酯和苯乙醇,且乙酸、己酸相对含量较高。各菌产可挥发性组分差异明显,相对含量较高的化合物种类为:短乳杆菌L5产烷烃类化合物(27.91%),铅黄肠球菌L8产醇类化合物(43.14%),鼠李糖乳杆菌L9产酯类(7.35%)、酮类(3.61%)和吡嗪类(3.3%)化合物,干酪乳杆菌L11产酸类(27.98%)和芳香类(5.96%)化合物。仅有短乳杆菌L5产四甲基吡嗪,短乳杆菌L5和铅黄肠球菌L8可明显产乙醇,鼠李糖乳杆菌L9和干酪乳杆菌L11产3-羟基-2-丁酮。这些物质对白酒风味和口感有重要影响。研究显示,窖泥中各种乳酸菌特征不一,对白酒酿造有不同影响。本研究旨在为进一步挖掘白酒酿造功能微生物、拓展乳酸菌的应用提供理论依据。     

The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml⁻¹ after10 days and 400 AU m1⁻¹ after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml⁻¹ were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.     

Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage.

A total of 26 Lactobacillus strains were isolated from various mild yoghurts and novel-type probiotic dairy products and from a starter culture preparation and were identified by using DNA-DNA hybridization technique. The species present in those products were found to be Lactobacillus acidophilus, L. johnsonii, L. crispatus, L. casei, L. paracasei and L. rhamnosus. DNA homology analysis revealed that some strains had been misclassified by their investigators. Three strains designated as L. acidophilus (L. acidophilus LA-1, L. acidophilus ATCC 43121 and the Lactobacillus strain from Biogarde culture) were found to belong to L. johnsonii and L. acidophilus L1 to be L. crispatus. Strains designated as L. casei were found to be members of three separate species: L. casei, L. paracasei and L. rhamnosus. Viable numbers of lactobacilli in mild and probiotic yoghurts varied greatly including some products with very low Lactobacillus counts. The majority of the probiotic yoghurts, however, contained viable counts above 10(5) per g even at the end of the best before use period.     

EVALUATION OF FUNCTIONAL ASPECTS IN LACTOBACILLUS STRAINS ISOLATED FROM DRY FERMENTED SAUSAGES

ABSTRACT

The aim of this study was to characterize lactobacilli isolated from a traditional Italian dry‐fermented sausages inoculated with Lactobacillus casei/paracasei for use as probiotics. Strains isolated were L. casei (38%), L. paracasei paracasei (32%), Lactobacillus rhamnosus (21%) and Lactobacillus sakei sakei (9%). L. paracasei paracasei and L. rhamnosus showed no significant decrease in viability compared with L. casei and L. sakei sakei in artificial gastric juice; L. casei and L. sakei sakei were not affected by artificial intestinal fluid. In human embryonic intestine cell line (INT407) in vitro model, L. rhamnosus and L. sakei sakei were more adhesive than the other strains. The production of acetic acid (40.92 ± 1.94 mmol/L) and propionic acid (0.14 ± 0.07 mmol/L) was similar in all strains tested. Considerably higher (100–250 mmol/L) levels of lactic acid were produced by L. casei, L. paracasei paracasei and L. rhamnosus, and were varied depending on microaerobic or anaerobic conditions. L. casei and L. rhamnosus had the best antibacterial activity against Escherichia coli and Salmonella enterica ssp. enterica (serovar Typhimurium). These findings suggest that L. casei and L. rhamnosus merit further investigation as novel starter cultures in meat products.

PRACTICAL APPLICATIONS

Probiotics are well established in dairy products but they are not widely used in meat products and it is expected their use in this area will expand considerably in the near future. Starter cultures of active or resting forms of microorganisms are widely employed in fermented meat products to improve sensory qualities. More recently they are being used in fermented meat products to improve the nutritional value of these products as functional foods. Probiotics in meat may exert their benefits by several methods, and more research is required to identify the strains and combinations of strains that produce the most health benefits. Our study demonstrates that the in vitro characterization of Lactobacillus strains directly isolated from dry fermented sausages can be useful to select novel starter cultures with potential probiotic characteristics among lactic acid bacteria used as commercial meat starter culture.

     

复合乳酸菌在发芽糙米乳中的发酵特性

将植物乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、干酪乳杆菌4种乳酸菌经三三组合后作为复合发酵剂对发芽糙米乳进行发酵,通过分析酸度、活菌数、脱水收缩作用敏感性、蛋白质分解力、流变学性质等实验结果,对各乳酸菌组合的发酵特性进行评价,最终筛选出适合发酵发芽糙米乳的最佳菌株组合。结果表明:植物乳杆菌-嗜酸乳杆菌-干酪乳杆菌组合所得到的发酵发芽糙米乳具有较高的品质。该发酵糙米乳在4℃条件下贮藏21 d后酸化程度较弱,下降了0.71个单位;活菌数变化小且冷藏期间数量一直高于8.7(lg(CFU/m L)),游离氨基酸平均含量达0.86 mmol/L,流变学性质显示其剪切稀化作用较弱。故该乳酸菌组合较为适合发酵发芽糙米乳。     

发酵型核桃乳生产专用乳杆菌的选育

从发酵面包、白芥丝、泡菜、酸菜中分离到14株乳杆菌,经核桃乳发酵产酸特性初筛,确定菌株L5、L7、L8及L12适合核桃乳发酵。通过菌落形态、菌体形态和16S rRNA基因序列分析,确定4株菌分别为鼠李糖乳杆菌（Lactobacillus rhamnosus）、副干酪乳杆菌（Lactobacillus paracasei）、食窦魏斯氏菌（Weissella cibaria）干酪乳杆菌（Lactobacillus casei）。将上述菌株与实验室保存的嗜热链球菌SA进行核桃乳发酵实验,感官评分分别为78分、95分、83分和82分;4个发酵核桃乳中9种氨基酸含量均有不同程度提高,其中菌株L7发酵产品的氨基酸含量增幅最大,由0.67%增至1.60%。因此副干酪乳杆菌L7更适合发酵型核桃乳的生产。     

乳杆菌发酵对黑米多酚含量及组成的影响

以植物乳杆菌（Lactobacillus plantarum 115,LP）、嗜酸乳杆菌（Lactobacillus acidophilus 14,LA）、干酪乳杆菌（Lactobacillus casei GB,LC）、保加利亚乳杆菌（Lactobacillus bulgaricus 134,LB）、副干酪乳杆菌（Lactobacillus paracasei 37,LPC）和鼠李糖乳杆菌（Lactobacillus rhamnosus GG,LGG）为发酵菌种,研究乳杆菌发酵对黑米游离酚和结合酚的含量及组成的影响。结果表明,6株乳杆菌发酵均显著提高了黑米游离酚的含量,但不同乳杆菌发酵后提高的程度具有显著差异,其中提高最显著的是LB。不同乳杆菌发酵后黑米中9种游离态和结合态单体酚的含量变化存在显著差异,其中,6株乳杆菌发酵后黑米中游离态的香草酸和对羟基苯甲酸的含量均显著降低（P<0.05）,LP菌株除外游离态咖啡酸的含量均显著提高（P<0.05）,而6株乳杆菌发酵后结合态阿魏酸含量均显著降低（P<0.05）。该研究为黑米的高效利用和天然活性产品的开发提供参考。     

Viability of commercial probiotic cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in cheddar cheese

Six batches of cheddar cheese were manufactured containing different combinations of commercially available probiotic cultures from three suppliers. Duplicate cheeses contained the organisms of each supplier, a Bifidobacterium spp. (each supplier), a Lactobacillus acidophilus (2 suppliers), and either Lactobacillus casei, Lactobacillus paracasei, or Lactobacillus rhamnosus. Using selective media, the different strains were assessed for viability during cheddar cheese maturation over 32 weeks. The Bifidobacterium sp. remained at high numbers with the three strains being present in cheese at 4 x 10(7), 1.4 x 10(8), and 5 x 10(8) CFU/g after 32 weeks. Similarly the L. casei (2 x 10(7) CFU/g), L. paracasei (1.6 x 10(7) CFU/g), and L. rhamnosus (9 x 10(8) CFU/g) strains survived well; however, the L. acidophilus strains performed poorly with both decreasing in a similar manner to be present at 3.6 x 10(3) CFU/g and 4.9 x 10(3) CFU/g after 32 weeks. This study indicates that cheddar cheese is a good vehicle for a variety of commercial probiotics but survival of L. acidophilus strains will need to be improved.