Reduced biogenic amine contents in sauerkraut via addition of selected lactic acid bacteria

The contents of free amino acids and biogenic amines in spontaneously fermented sauerkraut, inoculated or not with specific lactic acid bacterium strains, were monitored throughout 45 d of storage. The strains tested were Lactobacillus plantarum 2142, Lactobacillus casei subsp. casei 2763 and Lactobacillus curvatus 2771. In both the control and the experiments, the total amino acid contents increased with time – and the predominant ones were aspartic acid, arginine and glutamic acid. However, upon inoculation with either strain, the total biogenic amine contents remained considerably lower than those of the control (especially in the cases of L. casei subsp. casei and L. curvatus); every single biogenic amine was actually below the 100 ppm-threshold. The dominant biogenic amines in the control were putrescine – and tyramine and histamine, to a lesser extent; the putrescine content was 10-fold lower if inoculation had taken place with either lactobacillus tested; and histamine and tyramine were essentially absent during storage, whereas they ranked above 200 ppm in the control by 45 d. Hence, an efficient food-grade biological tool was made available that constrains buildup of dangerous biogenic amines in fermented vegetables during storage.     

Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage

Lactobacillus casei Zhang is a novel strain that was screened out of koumiss collected in Inner Mongolia, and our previous research showed that L. casei Zhang has health benefits such as cholesterol-reducing and immunomodulating effects. The fermentation characteristics of L. casei Zhang in soymilk and bovine milk and the transit tolerance of L. casei Zhang in fermented milk products during refrigerated storage for 28 d were assessed. A faster decrease in pH and faster growth of L. casei Zhang during fermentation were observed in soymilk compared with bovine milk at various inoculation rates, probably because of the low pH buffering capacity of soymilk. The fermented bovine milk samples had much higher final titratable acidity (TA) values (between 0.80 and 0.93%) than the soymilk samples (between 0.40 and 0.46%). Dramatic increases in TA values in the fermented soymilk samples during storage were observed, and the TA values of the fermented soymilk samples changed from <0.56% to values between 0.86 and 0.98%. On the other hand, only slight increases in TA were observed in the bovine milk samples during the 28 d of storage. The survival rates of freshly prepared cultures of L. casei Zhang in simulated gastric juice at pH 2.0 and 2.5 were 31 and 69%, respectively, and the delivery of L. casei Zhang through fermented soymilk and bovine milk significantly improved the viability of L. casei Zhang in simulated gastric transit. Lactobacillus casei Zhang showed good tolerance to simulated gastric juice and intestinal juice in the fermented soymilk and bovine milk samples, and maintained high viability (>10⁸ cfu/g) during storage at 4°C for 28 d. Our results indicated that both soymilk and bovine milk could serve as vehicles for delivery of probiotic L. casei Zhang, and further research is needed to elucidate the mechanism of the change in pH and TA of L. casei Zhang in fermented milk samples during fermentation and storage and to understand the difference between soy- and milk-based systems.     

Effect of addition of inulin and galactooligosaccharide on the survival of microencapsulated probiotics in alginate beads coated with chitosan in simulated digestive system,yogurt and fruit juice

Galactooligosaccharides (GOS) and inulin were added during microencapsulation of Lactobacillus acidophilus 5 and Lactobacillus casei 01 in alginate beads coated with chitosan at the concentrations of 0, 0.5, 1.0 and 1.5%. Addition of prebiotics significantly (p < 0.05) increased the bead size by approximately 3.8%. The presence of GOS (0.3%) in the microencapsulation provided the best protection with only 3.1 and 2.9 logs reduction for L. acidophilus 5 and L. casei 01, respectively, after incubation in simulated gastric juice (pH 1.55), followed by simulate intestinal juice containing 0.6% bile salt. The viabilities of microencapsulated probiotics containing 1.5% GOS in commercial yogurt and orange juice were also performed at refrigerated storage for 4 weeks. In yogurt, the numbers of cells with GOS were higher than those of without GOS by approximately 1.1 and 0.4 logs for L. acidophilus 5 and L. casei 01, respectively. In orange juice, the numbers of cells with GOS were higher than those of without GOS by approximately 0.5 and 0.4 logs for L. acidophilus 5 and L. casei 01, respectively. The numbers of probiotic bacteria were maintained above the recommended therapeutic minimum (10⁷ cfu g⁻¹ or mL⁻¹ of product) throughout the storage in both products.     

Yerba-mate (Ilex paraguariensis) extract prevents ethanol-induced liver injury in rats

Ethanol metabolism-associated oxidative stress contributes to the pathogenesis of alcoholic liver disease. We examined the effect of a Mate tea extract on ethanol-induced liver injury in vitro and in vivo models. Isolated hepatocytes were incubated with ethanol. An extract of Yerba-Mate tea (EMT) was added to the cultures simultaneously with ethanol. EMT treatment suppressed the ethanol-induced increase in cell death by inhibiting cytochrome p450 2E1 (CYP2E1) activity, which is related to the production of reactive oxygen species. Furthermore, we examined the effects of EMT on serum transaminase activity, and the progression of liver fibrosis in rats treated with ethanol and CCl₄. Rats were fed a diet that included 0.005% or 0.02% EMT or no EMT. For a period of 3 weeks, the animals were provided drinking water containing 5% ethanol and were also treated with carbon tetrachloride (CCl₄) (0.1 ml/kg of body weight). EMT treatment suppressed plasma ALT and AST activities in the ethanol- and CCl₄-treated rats. EMT treatment also decreased CYP2E1 expression and increased ADH expression in the ethanol- and CCl₄-treated rats. EMT treatment fully protected the rats against ethanol- and CCl₄-induced liver injury. These results suggest that EMT may serve as a candidate for preventing ethanol-induced liver injury.     

Enumeration of Lactobacillus casei in the presence of L. acidophilus,bifidobacteria and lactic starter bacteria in fermented dairy products

The aim of this work was to select a set of culture media to perform the enumeration of L. casei when it appears together with L. acidophilus, bifidobacteria and bacteria of lactic acid starters, in fermented dairy products. A number of L. acidophilus, Bifidobacterium and L. casei strains (8 of each) were tested for their ability to grow in two selective/differential media (LP-MRS agar and B-MRS agar). The enumeration of these bacteria using the media mentioned above was also carried out in fermented dairy products available on the market. B-MRS agar and LP-MRS agar were able to inhibit the lactic acid bacteria from the starters. B-MRS agar could be useful for the selective colony count of L. acidophilus or L. casei, or for a differential enumeration when these bacteria appear together in a fermented dairy product. In the case of bifidobacteria, a selective colony count could be performed on LP-MRS agar, even when it appears with L. casei, because a differential cell enumeration between both organisms was possible. The three probiotic bacteria could be simultaneously enumerated from dairy products that contain them along with starter bacteria using media developed in this work.     

Addition of probiotic bacteria in a semi-hard goat cheese (coalho): Survival to simulated gastrointestinal conditions and inhibitory effect against pathogenic bacteria

In this study, the survival of the probiotics Lactobacillus acidophilus (LA-5), Lactobacillus casei subsp. paracasei (L. casei 01) and Bifidobacterium lactis (BB12) incorporated in a Brazilian semi-hard goat cheese (coalho) when exposed to in vitro simulated conditions of digestion was assessed. The inhibitory effects of these probiotic bacteria were also evaluated against Listeria monocytogenes and Staphylococcus aureus in the goat coalho cheese during refrigerated storage. At the end of the in vitro digestion, all of the probiotic tested strains presented decreased (p < 0.05) viable cell counts (5.5–6.0 log cfu/g) with respect to those determined before exposure to the mouth conditions (7–8 log cfu/g). L. casei subsp. paracasei presented inhibition rate of 7.87% and 23.63% against S. aureus on the 14th and 21st day of storage at 10 °C, respectively; against L. monocytogenes these values were 12.96 and 32.99%. Positive inhibition rates of B. lactis toward S. aureus were found on the 1st, 14th and 21st days of storage (16.32%, 10.12% and 3.67%, respectively); and against L. monocytogenes only on the 1st day of storage (3.28%). From these results, goat coalho cheese could be an interesting carrier of probiotic strains of L. acidophilus, L. casei subsp. paracasei and B. lactis. Moreover, L. casei subsp. paracasei, could be used as protective culture for delaying the growth of S. aureus and L. monocytogenes in goat coalho cheese.     

Identification of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis

A rapid and reliable technique for identifying the strains of lactic acid bacteria (LAB) was developed in this study, and the 16S ribosomal RNA gene (16S rRNA gene) was used as the target. Several species-specific primer pairs were designed based on the variability of 16S rRNA sequence(s) for differentiating 5 strains of lactobacilli which were added into probiotic products in Taiwan. It was simple to identify Lactobacillus acidophilus and L. delbrueckii by species-specific primers, but it could not be used to distinguish L. casei, L. paracasei and L. rhamnosus. Another PCR approach was developed with hybridization probes which were designed according to the difference among the 16S rRNA genes of L. casei, L. paracasei and L. rhamnosus, and melting curve analysis of the hybridization probe was used to distinguish them. It was found that this approach could identify L. paracasei and L. rhamnosus correctly but not separate L. paracasei from L. casei, the result was due to both of them had the same 16S rRNA sequence. These results suggest that melting curve analysis of PCR approach in this study is a rapid, simple and accurate method in distinguishing the closely related strains of lactobacilli.     

Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress

Tolerance to acid is an important feature for probiotic bacteria during transition through the gastrointestinal tract. Proteomics analysis of a new probiotic bacterium, Lactobacillus casei Zhang, was performed upon 30-min exposure to low acid stress (pH 2.5 vs. pH 6.4) using two-dimensional electrophoresis. Out of 33 protein spots that showed changes of expression between the two pHs, 22 showed 1.5-fold higher expression at pH 2.5 than at pH 6.4, whereas five spots had expression decreased by 1.5-fold at pH 2.5. There were also six protein spots that were exclusively present on different pH maps. Further analysis showed that eight of the enhanced proteins, NagA, NagB, PGM, GlmM, LacC, TDP, GALM and PtsI, were involved in carbohydrate catabolism. Moreover, quantitative RT-PCR showed that the mRNA expression levels of dnaK, nagB, galm, estC, tuf and luxS were consistent with changes in protein expression. We postulate that there might be some relationship between differentially expressed proteins and acid tolerance in L. casei Zhang.     

Effect of alsD deletion and overexpression of nox and alsS on diacetyl and acetoin production by Lacticaseibacillus casei during milk fermentation

Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.     

Competition mechanisms of lactic acid bacteria and bifidobacteria: Fermentative metabolism and colonization

Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.     

The physicochemical surface characteristics of Lactobacillus casei

The surface and interfacial properties of five strains of Lactobacillus casei, commonly found in cheese, were investigated in an attempt to identify surface properties that might be exploited to effect adsorption of the cells onto milk fat globules. All measures of surface hydrophobicity revealed these strains to be moderately hydrophobic compared to a reference strain of Mycobacterium smegmatis which was very hydrophobic. In chromatography experiments, L. casei had no affinity for either hydrophobic (octyl-sepharose) or control (sepharose) columns in conditions of physiological pH and low ionic strength, despite a favorable free energy of interfacial interaction (ΔG^IF_IW2) as derived from contact angle measurements. Adhesion of L. casei strains to these materials was effected by manipulation of eluent pH and ionic strength. In contrast, adhesion of L. casei strains to n-hexadecane concurred with ΔG^IF_IW2. Surfaces of L. casei strains subjected to X-ray photoelectron spectroscopy exhibited similar amounts of carbon and oxygen but displayed considerable diversity in surface nitrogen and phosphorous contents. Carbon existed predominantly as C-(O, N) and oxygen as -OH on L. casei surfaces but carbon on M. smegmatis surfaces was present predominantly as C-(C,H).     

Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.     

Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one strains of lactic acid bacteria (LAB) isolated from Armada cheese, Sobado variety, (eight strains of Lactococcus lactis subsp. lactis, four strains of Lactococcus lactis subsp. cremoris, two strains of L. lactis subsp. lactis biovar. diacetylactis, two strains of Leuconostoc mesenteroides subsp. mesenteroides, two strains of Leuconostoc mesenteroides subsp. dextranicum, five strains of Lactobacillus plantarum, six strains of Lactobacillus casei subsp. casei and two strains of Lactobacillus brevis) were screened for their acidifying capacity and enzymatic activity, that included the rapid API-ZYM system, the proteolytic activity, the amino-, di-, and carboxypeptidase activity and the caseinolytic activity. The strains of L. lactis subsp. lactis exhibited the highest acidifying and proteolytic activity. Lipase and esterase activity was practically non-existent for lactococci and lactobacilli; a certain esterase activity was observed among leuconostoc. The highest aminopeptidase activity was demonstrated by the cell-free extract (CFE) of some strains of L. plantarum, L. casei subsp. casei and L. mesenteroides subsp. dextranicum. The CFEs of L. lactis subsp. cremoris and L. lactis subsp. lactis possessed carboxypeptidase and dipeptidase activities, at levels depending on the strain. Appreciable caseinolytic activity was detected for the CFE of L. plantarum and those some lactococci.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

The growth behaviour and enhancement of probiotic viability in bioyoghurt

The growth behaviour of Lactobacillus casei-01 and Bifidobacterium bifidum Bb-12 in bioyoghurt made with three different yoghurt starter cultures (YC-Fast 1, YC-380 and YC-180) and the enhancement of probiotic viability, were investigated. The titratable acidity (TA) increased rapidly in yoghurt samples made with YC-Fast 1. The fermentation times were 3, 3.5 and 4 h for bioyoghurts made with YC-Fast 1, YC-380 and YC-180, respectively. The total viable counts of L. casei and B. bifidum were the highest in bioyoghurt samples made with YC-180. Microencapsulation of L. casei and B. bifidum enhanced their viability and this technique can be used with normal yoghurt starter. Also, utilization of heat shock yoghurt starter enhanced the viability of probiotics.     

CRISPR-Cas9–mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate–induced inflammation in bovine mammary epithelial cells

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats–Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.     

Short communication: Effect of milk and milk containing Lactobacillus casei on the intestinal microbiota of mice

BALB/c mice were fed milk or Lactobacillus casei BL23 in milk for 14 d and fecal samples were collected at d 0, 4, and 7 as well as 1 and 8 d after the last administration. According to high-throughput DNA sequencing of the 16S rRNA genes extracted from the fecal microbiota, the bacterial diversity in the fecal samples of all mice increased over time. After 14 d of administration, the consumption of milk and milk containing L. casei BL23 resulted in distinct effects on the microbial composition in the intestine. Specifically, the proportions of bacteria in the Lactobacillaceae, Porphyromonadaceae, and Comamonadaceae were significantly higher in mice fed the L. casei BL23-milk culture compared with one or more of the other groups of mice. The relative amounts of Lachnospiraceae were higher and Streptococcaceae were lower in mice fed milk alone. The changes were not found at d 4 and 7 during milk and L. casei feeding and were no longer detected 8 d after administration was stopped. This study shows that consumption of milk or probiotic L. casei-containing milk results in non-overlapping, taxa-specific effects on the bacteria in the distal murine intestine.