Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus

Recognized to confer health benefits to consumers, probiotics such as Lactobacillus acidophilus are commonly incorporated into fermented dairy products worldwide; among which yogurt is a popular delivery vehicle. To materialize most of the putative health benefits associated with probiotics, an adequate amount of viable cells must be delivered at the time of consumption. However, the loss in their viabilities during refrigerated storage has been demonstrated previously. This study focused on the effects of yogurt starter cultures on the survival of five strains of L. acidophilus, with emphases on low pH and acid production. Differential survival behavior between L. acidophilus strains was further analyzed. To this end, viable cell counts of L. acidophilus were determined weekly during 4 °C storage in various types of yogurts made with Streptococcus thermophilus alone, L. delbrueckii ssp. bulgaricus alone, both species of the starter cultures, or glucono-delta-lactone (GDL). All yogurt types, except for pasteurized yogurts, were co-fermented with L. acidophilus. Yogurt filtrate was analyzed for the presence of any inhibitory substance and for the amount of hydrogen peroxide. Multiplication of L. acidophilus was not affected by the starter cultures as all strains reached high level on day 0 of the storage period. Throughout the 28-day storage period, cell counts of L. acidophilus PIM703 and SBT2062 remained steady (~ 6 × 10⁷ CFU/g) in yogurts made with both starter cultures, whereas those of ATCC 700396 and NCFM were reduced by a maximum of 3 and 4.6 logs, respectively. When starter cultures were replaced by GDL, all strains survived well, suggesting that a low pH was not a critical factor dictating their survival. In addition, the filtrate collected from yogurts made with starter cultures appeared to have higher inhibitory activities against L. acidophilus than that made with GDL. The presence of viable starter cultures was necessary to adversely affect the survival of some strains, as pasteurized yogurts had no effect on their survival. In particular, the inhibitory effect exerted by L. delbrueckii ssp. bulgaricus on L. acidophilus NCFM was highly pronounced than by S. thermophilus, nevertheless, the same effect was not observed on SBT2062. The inhibition against stationary-phase NCFM cells might be caused by the elevated level of hydrogen peroxide produced by L. delbrueckii ssp. bulgaricus. Delineating factors driving the differences in survival trait among probiotic strains will lead to a more efficacious delivery of health benefits in fermented dairy products through targeted technological interventions.     

Study on the Influence of Tea Extract on Probiotics in Skim Milk: From Probiotics Propagation to Metabolite

In this study, the influence of tea extract (TE) on the growth of probiotics in skim milk was examined. Lactobacillus plantarum ST‐III, Bifidobacterium bifidum Bb02, Lactobacillus acidophilus NCFM, and Lactobacillus rhamnosus GG were used in this study. The introduction of TE in milk significantly stimulated the propagation and acidification of L. rhamnosus GG and L. acidophilus NCFM. The antioxidant capacities and the total free amino acid contents of all fermented milk products were enhanced by the addition of TE; however, there were different antioxidant properties and free amino acid contents of fermented milk samples fermented by different bacteria. With a 9% (w/w) level, the fermentation with L. rhamnosus GG and L. acidophilus NCFM showed larger numbers of viable cells and faster acidifying rates, as well as excellent antioxidant capacity and abundant free amino acids.  The stimulative effects of TE on probiotics can be considered for industrial purposes and has practical implications for commercial applications.     

Human milk oligosaccharide consumption by probiotic and human-associated bifidobacteria and lactobacilli

Human milk contains high concentrations of nondigestible complex oligosaccharides (human milk oligosaccharides; HMO) that reach the colon and are subsequently fermented by the infant gut microbiota. Using a high-throughput, low-volume growth determination, we evaluated the ability of 12 lactobacilli and 12 bifidobacteria strains, including several commercial probiotics, to ferment HMO and their constituent monomers. Of the 24 strains tested, only Bifidobacterium longum ssp. infantis ATCC 15697 and Bifidobacterium infantis M-63 were able to ferment 3′-sialyllactose, 6′-sialyllactose, 2′-fucosyllactose, and 3′-fucosyllactose. Bifidobacterium infantis M-63 degraded almost 90% of the 2′-fucosyllactose but left most of the fucose in the supernatant, as detected by HPLC. Among bifidobacteria, only the B. infantis strains and Bifidobacterium breve ATCC 15700 were able to ferment lacto-N-neotetraose (LNnT). Among lactobacilli, Lactobacillus acidophilus NCFM was found to be the most efficient at utilizing LNnT. The extracellular β-galactosidase (lacL, LBA1467) of L. acidophilus NCFM cleaves the terminal galactose of LNnT for growth, leaving lacto-N-triose II in the media as detected by HPLC. Inactivation of lacL abolishes growth of L. acidophilus NCFM on LNnT. These results contribute to our knowledge of HMO–microbe interactions and demonstrate the potential for synbiotic combinations of pre- and probiotics.     

Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng

Traditional Chinese medicinal herb Salvia miltiorrhiza (SM) and Panax notoginseng (PN) have been widely used for the prevention and treatment of vascular diseases in the clinics. To better understand their mechanisms of pharmacological actions, the in vitro antioxidant activities of extract of Salvia miltiorrhiza (ESM) and extract of Panax notoginseng (EPN) were evaluated with different antioxidant testing systems. Their activities of scavenging superoxide anion radicals, DPPH radicals, hydroxyl radicals, and hydrogen peroxide, chelating Ferrous ion, and ferric ion reducing power were assessed. The results showed that the mechanisms of their antioxidant effects were distinct and diverse. ESM possessed strong reducing power and high scavenging activities against free radicals including superoxide anion, hydroxyl and DPPH radicals, but a weaker scavenging activity for hydrogen peroxide. ferrous ion chelating activity of ESM was undetectable at the tested concentrations. In contrary, EPN exhibited strong ferrous ion chelating activity and high scavenging activities against hydrogen peroxide, hydroxyl radicals, and a weak activity against superoxide anion and DPPH free radicals. EPN did not show any ferric ion reducing power. Since their antioxidant mechanisms are complementary, the combined use of ESM and EPN might be even more beneficial. These antioxidant properties of SM and PN are likely part of the reasons that they are effective in the prevention and treatment of vascular diseases.     

Isolation of exopolysaccharide producing Lactobacillus strains from sorghum distillery residues pickled cabbage and their antioxidant properties

Twelve Lactobacillus strains isolated from sorghum distillery residue (SDR) pickled cabbage were studied for their in vitro scavenging activity against hydroxyl radicals and DPPH free radicals, inhibition of lipid peroxidation, and their resistance to hydrogen peroxide in cell lysate or intact cells. Lactobacillus brevis D7, at a dose of 10¹⁰ CFU/mL, showed the highest hydroxyl radical and DPPH scavenging activities, as well as total antioxidative activity, with inhibition rates of 51.2, 44.9, and 65.2%, respectively in intact cells. On the other hand, the L. brevis D7 strain was the most resistant against hydrogen peroxide. L. brevis D7 isolated from SDR pickled cabbage should be considered as a potential antioxidant to be used in functional foods. Correlation analysis showed that DPPH free radical scavenging activity is a potential screening indicator for high exopolysaccharide (EPS) producing Lactobacillus species.     

猕猴桃根提取物体外抗氧化作用的研究

探讨猕猴桃根提取物的体外抗氧化作用.采用DPPH自由基、超氧阴离子、羟基自由基、过氧化氢和还原力的反应体系,测定猕猴桃根提取物的体外抗氧化作用,并用VC进行对照实验.实验条件下,ERHM对DPPH自由基、超氧阴离子(·O2-)、羟基自由基(·OH)、过氧化氢H2O2等均有较强的清除或抑制作用,且显示较好的量效关系,同时具有一定的还原力.其消除DPPH自由基的EC50为8.03 μg/mL,清除超氧阴离子(·O2-)的EC5o为1.28 mg/mL,抑制羟基自由基(·OH)能力可达69.4％,浓度为100 μg/mL时对过氧化氢(H2O2)的清除率为42％.猕猴桃根提取物具有较强的还原力,能有效清除DPPH和超氧阴离子自由基,并抑制羟基自由基的产生.所以,ERHM有效成分具有较为显著的抗氧化作用.     

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.     

Synergy effects by combination with lactic acid bacteria and frutooligosaccharides on the cell growth and antimicrobial activity

The aim of this study was to identify probiotic possibility of lactic acid bacteria (LAB) from mustard leaf kimchi and investigate synergy effects of combination with LAB and fructooligosaccharide (FOS) on antimicrobial activity. Lactobacillus acidophilus GK20, L. paracasei GK74, and L. plantarum GK81 have high survival ratio under gastric and intestinal fluids and were resistant to ampicillin, streptomycin, or tetracycline. The pH of LAB gradually decreased but the titratable acidity increased owing to organic acids production. Especially, L. acidophilus GK20 and L. paracasei GK74 produced hydrogen peroxide and bacteriocin. When LAB was co-cultured with Listeria monocytogenes or Staphylococcus aureus in broth without FOS, the inhibition ratio of L. monocytogenes and S. aureus by L. acidophilus GK20 was 36 and 57%, but L. paracasei GK74 showed higher inhibitory effect against L. monocytogenes than S. aureus. Meanwhile, the cell growth, culture pH, titratable acidity, and bacteriocin activity of L. acidophilus GK20 and L. paracasei GK74 were significantly (p<0.05) increased in MRS with FOS than without prebiotic. In conclusion, L. acidophilus GK20 and L. paracasei GK74 could be potentially used as novel probiotic strains, moreover, the combination between L. acidophilus GK20 or L. paracasei GK74 and FOS has great potential as a synbiotic.     

Antioxidative and prooxidative effects of extracts made from cherry liqueur pomace

The effect of phenolics extracted from cherry liqueur pomace on the Fenton reaction was tested in a model system, where hydroxyl radicals from iron (II) reduction of H₂O₂ reacted with ethanol, forming 1-hydroxyl radicals. The extent of the radical formation was monitored by electron spin resonance (ESR) detection of spin adducts formed from 1-hydroxyethyl radicals and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN). Small phenol–iron ratios gave low amounts of detected radicals, indicating an antioxidative effect of the extracts, whereas high phenol–iron ratios gave high levels of radicals, indicating decreasing antioxidant or dominating prooxidant effects. Extracts made with pure solvents (water, methanol, ethanol, acetone or 2-propanol) gave higher antioxidant effects than extracts made with 70% aqueous solvent mixtures. The prooxidant effect of the extracts increased with the polarity of the extraction solvent. Gallic acid gave a prooxidative effect, p-coumaric acid only an antioxidative effect, while epicatechin gave an antioxidative effect at low concentrations and a prooxidative effect at high concentrations.     

Antioxidant properties of herbal extracts selected from screening for potent scavenging activity against superoxide anions

BACKGROUND: In a previous study with the electron spin resonance (ESR) spin‐trapping method on large‐scale screening with ethanol extracts of approximately 1000 kinds of herbs, four herbal extracts with prominently potent ability to scavenge superoxide anions were chosen, namely the extracts from Punica granatum (peel), Syzygium aromaticum (bud), Mangifera indica (kernel) and Phyllanthus emblica (fruit)). In the present study, these extracts were further examined to determine if they also scavenge hydroxyl radicals, by applying the ESR spin‐trapping method, and if they have heat resistance as a desirable characteristic feature. RESULTS: Experiments with the Fenton reaction and UV irradiation of hydrogen peroxide, both of which generate hydroxyl radicals, showed that all four extracts had potent ability to directly scavenge hydroxyl radicals. Each extract exerted more potent hydroxyl radical‐scavenging activity than mannitol and salicylic acid. Furthermore, the scavenging activities against superoxide anions and hydroxyl radicals of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat‐resistant. CONCLUSION: The four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, three of them are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance. Copyright © 2008 Society of Chemical Industry     

Short communication: Salt tolerance of Lactococcus lactis R-604 as influenced by mild stresses from ethanol,heat, hydrogen peroxide,and UV light

Lactococcus lactis is a culture widely used in salt-containing dairy products. Salt hinders bacterial growth, but exposure to environmental stress may protect cells against subsequent stress, including salt. The objective of this study was to evaluate the salt tolerance of L. lactis R-604 after exposure to various stresses. The culture was subjected to 10% (vol/vol) ethanol for 30 min, mild heat at 52°C for 30 min, 15 mM hydrogen peroxide for 30 min, or UV light (254 nm) for 5 min and compared with a control. Starting with 5 log cfu/mL for all treatments, growth was determined in M17 broth with 5 NaCl concentrations (0, 1, 3, 5, and 7% wt/vol). Plating was conducted daily for 5 d. Salt tolerance was enhanced with mild heat exposure before growth in M17 broth with 5% (wt/vol) NaCl on d 3, 4, and 5, and with exposure to hydrogen peroxide and ethanol stresses before growth in M17 broth with 5% (wt/vol) NaCl on d 4 and 5. Exposure of this culture to mild heat, hydrogen peroxide, or ethanol before growth in M17 broth containing 5% (wt/vol) salt can enhance its survival, which could be beneficial when using it in salt-containing dairy products.     

IN VITRO ANTIOXIDANT ACTIVITIES OF WATER-SOLUBLE POLYSACCHARIDES EXTRACTED FROM ISARIA FARINOSA B05

The water‐soluble exo‐polysaccharides (WSEPS) and intrapolysaccharides (WSIPS) were prepared from the fermentation broth and mycelia of Isaria farinosa B05, respectively, and their antioxidant activities in vitro were studied by various antioxidant assays, including hydroxyl radical scavenging, superoxide radical scavenging, hydrogen peroxide (H₂O₂) scavenging, metal chelating activities, reducing power and antilipid peroxidation. The results show that the water‐soluble polysaccharides extracted from I. farinosa B05 had a high antioxidant activity in a concentration‐dependent manner (except the activity of antilipid peroxidation). WSIPS quenched hydroxyl radicals at high amounts but not at low amounts; WSEPS did not scavenge hydroxyl radicals. WSEPS and WSIPS had a similar activity in scavenging superoxide radicals. WSIPS had a slightly higher H₂O₂‐scavenging activity than WSEPS before the activity reached 100% at the amount of 10.24 mg. Similar to the result of hydroxyl radical scavenging, WSIPS, but not WSEPS, chelated Fe²⁺. The reductive ability of WSEPS and WSIPS was similar and significant compared with the control at the range of 0.8–6.4 mg. Both WSEPS and WSIPS showed higher antilipid peroxidation activities at low amounts (≤ 0.64 mg) than at high amounts. These results indicate that WSEPS and WSIPS had strong antioxidant activities and can be explored as novel potential antioxidants.     

Iron-chelating ability and antioxidant properties of phycocyanin isolated from a protean extract of Spirulinaplatensis

The invitro scavenger activities of different reactive oxygen species (superoxide radical, hydroxyl radical, hydrogen peroxide, hypochlorous acid and peroxyl radical), the effects on lipid peroxidation and the iron-chelating ability of a Spirulinaplatensis protean extract and the biliprotein, phycocyanin, isolated from this microalga were studied. S. platensis protean extract inhibited the generation of hydroxyl radical (IC₅₀ = 537 μg/ml for the system with EDTA and 1500 μg/ml without EDTA), the production of peroxyl radical (IC₅₀ = 230 μg/ml), and the lipid peroxidation process (IC₅₀ = 2320 μg/ml for the enzymatic system and 2180 μg/ml for the non-enzymatic system). Besides, phycocyanin inhibited hydroxyl and peroxyl radicals and the lipid peroxidation process. The iron ions decreased the maximum fluorescence emission spectra of S. platensis protean extract and phycocyanin and it was an indicator of the metal-chelating activity. The antioxidant properties of S. platensis and phycocyanin may arise from both radical-scavenging and metal chelation. Our results suggest that S. platensis could be used as a dietary supplement to prevent some diseases where free radicals are involved.     

Effect of incubation conditions and possible intestinal nutrients on cis-9, trans-11 conjugated linoleic acid production by Lactobacillus acidophilus F0221

The purpose of this study was to optimise the incubation conditions for cis-9, trans-11 conjugated linoleic acid (c9, t11 CLA) production by Lactobacillus acidophilus F0221 and evaluate the effect of possible intestinal nutrients on c9, t11 CLA production. Growth in Mann Rogosa Sharp broth supplemented with cysteine and containing 0.5 g L⁻¹ linoleic acid at pH 6.5 with anaerobic incubation at 37 °C for 40 h were found to be the optimal conditions for CLA production. Galactosaccharide, arabinogalactan, galactose and glucose had a higher promoting effect on CLA production (96.19–123.89 μg mL⁻¹) than other carbohydrates. Acetic acid had a higher promoting effect on CLA production (130.98 μg mL⁻¹) than other short chain fatty acids. These results provide detailed parameters for the production of c9, t11 CLA by L. acidophilus F0221 and are valuable for further understanding of c9, t11 CLA formation in the human intestine by this strain.     

Antioxidant and DNA Damage Protection Activities of Duck Gizzard Peptides by Chemiluminescence Method

Antioxidant and DNA damage protection activity of duck gizzard peptides were evaluated using chemiluminescence method. The duck gizzard peptides exhibited free radical scavenging activity against superoxide anions, hydroxide radicals, and hydrogen peroxide, as well as DNA damage protection in a dose-dependent manner. The duck gizzard peptides had varying degrees of antioxidant activity for the inhibition of different radical species as well as DNA damage, with half-inhibition concentration (IC₅₀) values of 14.58, 0.0163, 19.80, and 1.93 mg/mL, respectively. The duck gizzard peptides had the strongest inhibition of hydroxyl radical activity, and the weakest inhibition of hydrogen peroxide radicals. The results clearly indicated that duck gizzard peptides were good sources of natural antioxidants for future applications in the food industry.     

Evaluation of antioxidant activities of various solvent extracts of Carissa opaca fruits

The chloroform and aqueous fractions of Carissa opaca fruit, a traditional medicinal fruit in Pakistan possessed a high amount of total phenolic and flavonoid contents as compare to other solvent fractions with potent antioxidant activities in scavenging DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radicals, and had strong iron chelating activity. On the other hand, the ethyl acetate fraction showed the highest inhibition of β-carotene linoleic acid peroxidation and phosphomolybdate assay. A high correlation coefficient existed between EC₅₀ values of DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radicals, total phenolics and flavonoids, but a non significant correlation was found in the case of iron chelaters, β-carotene and phosphomolybdate assay. This study verified that the chloroform and aqueous fractions have strong antioxidant activities which were correlated with its high level of phenolics and flavonoids. These fractions can be used as a source of potential antioxidant or functional food material.     

Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk

Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus NCFM cells propagated in 11% skim milk during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 open reading frames were differentially expressed at least in one time point. Genes differentially expressed in skim milk included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4 h, whereas gene expression of Opp2 increased over time reaching its highest level at 12 h, suggesting that the 2 systems have different specificities. Expression of a 2-component regulatory system, previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (autoinducer-2) and adherence to mucin and intestinal epithelial cells, in vitro.     

Antioxidant properties of Cortex Fraxini and its simple coumarins

We present the antioxidant properties of crude extract, fractions and ingredients of Cortex Fraxini (CF) and compare the antioxidant capacities of coumarin ingredients of CF and known antioxidants, including catechin, quercetin, ascorbic acid, trolox and BHT. The IC₅₀ values for CF in the DPPH and TEAC methods were 406 and 39.3 μg/ml, respectively. Among all fractions the chloroform fraction is the most active fraction in scavenging DPPH, ABTS and hydroxyl radicals, and there is a significant relationship between the antioxidant activities and the contents of the antioxidant phenolic ingredients. The contents of esculetin and fraxetin in the chloroform fraction were 8.44% and 11.1%, respectively. Esculetin and fraxetin also had good radical-scavenging capacities, and esculetin was the best, among all test compounds, against the DPPH radical. Moreover, esculetin and fraxetin had selective scavenging activity upon hydroxyl radicals and hydrogen peroxide, and this potency was better than known antioxidants and equal to quercetin in scavenging hydrogen peroxide. These results show that CF, partitioned with chloroform, gave a phenolic coumarin-enriched fraction, and that this fraction had the best free radical-scavenging activity and inhibition of Fe²⁺/ascorbate-induced lipid peroxidation, mainly due to its reducing power.