Protective activity of probiotic bacteria against 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) – an in vitro study

Heterocyclic aromatic amines (HAAs) are carcinogenic compounds present in a typical Western diet rich in thermally processed meat. These nutritional factors can modulate the cytotoxicity of faecal water (FW) and induce tumours in the human gastrointestinal tract. Supplementation with probiotics is promising in terms of reducing the harmful effects of HAAs in the human body. The aim of the study was in vitro assessment of the protective activity of the probiotic strains Lb. rhamnosus 0900, Lb. rhamnosus 0908, Lb. casei 0919 and Lb. casei DN 114001 against IQ (2-amino-3-methyl-3H-imidazo[4,5-f]quinoline) and PhIP (2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine) after incubation with faeces from 15 persons aged 4 months to 82 years (children, adults and the elderly). The highest mean cytotoxicity of FW was observed for the elderly (63.2% ± 3.7%) and the lowest for children (28.0% ± 9.5%), as estimated by a neutral red uptake assay. The probiotics lowered the average cytotoxicity of FW exposed to IQ or PhIP. The concentration of IQ and PhIP in FW was most effectively reduced by Lb. rhamnosus 0900 (47.5%) and Lb. casei 0919 (45.8%), respectively, as determined by high -performance liquid chromatography. All the tested strains bound PhIP to a higher extent than IQ. In an alkaline comet assay, Lb. casei 0919 and Lb. rhamnosus 0908 displayed the strongest protective effect against IQ and PhIP (up to 80% reduction of DNA damage). Also in a comet assay, Lb. rhamnosus 0908 exhibited antioxidative activity toward H₂O₂ and PhIP (up to 63% and 69.5% reduction of oxidative DNA damage, respectively). The protective activity of the probiotic strains was specific to a given person’s FW, which implies the involvement of intestinal microbiota in the process.     

Effect of gums on viability and β‐galactosidase activity of Lactobacillus spp. in milk drink during refrigerated storage

The viability and β‐galactosidase activity of four Lactobacillus strains in milk drink containing gums during 28 days of refrigerated storage at 4 °C were assessed. The population of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were maintained, whereas the population of Lactobacillus reuteri DSM20016 and Lactobacillus reuteri SD2112 significantly decreased. The recommended level of 6 log CFU g^?1 was exceeded for all tested trains throughout storage. The highest viable number of Lactobacillus rhamnosus GGB103 (8.76 ± 0.03 log CFU mL^?1) was obtained in the product containing carrageenan–maltodextrin. The addition of guar–locust bean–carrageenan led to 20‐fold increase in the level of β‐galactosidase activity for L. rhamnosus GGB101 (1208 ± 2.12 Miller units mL^?1) compared to the control (61 ± 2.83 Miller units mL^?1). Our results suggested that gums could be added to milk to improve viability and enhance β‐galactosidase activity of Lactobacillus.     

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.

     

Effect of a bacteriocin‐producing strain of Lactobacillus paracasei on the nonstarter microflora of Cheddar cheese

In this study, the growth of chloramphenicol‐resistant bacteriocin‐sensitive indicator strain Lactobacillus casei DPC 2048^CM was evaluated in Cheddar cheese made with bacteriocin‐producing Lactobacillus paracasei DPC 4715. No suppression of growth of the indicator strain was observed in the cheese during ripening, and no bacteriocin production by L. paracasei DPC 4715 was detected by the well diffusion method in cheese and cheese extracts. The bacteriocin produced by L. paracasei DPC 4715 was sensitive to chymosin and cathepsin D, and it may have been hydrolysed by the rennet used for cheese manufacture or by indigenous milk proteases.     

Adherence of probiotic bacteria to human colon epithelial cells and inhibitory effect against enteric pathogens – In vitro study

The aim of this study was to determine the anti‐adherence properties of three probiotic lactobacillus strains (Lb. rhamnosus 0900, Lb. rhamnosus 0908 and Lb. casei 0919), and their mixture against pathogens: Escherichia coli ATCC 10536, Salmonella enterica serovar Typhimurium ATCC 14028 and Candida albicans ATCC 10231 using Caco‐2 human colon adenocarcinoma cells. All strains of lactobacilli and the probiotic mixture to the greatest extent inhibited adherence of S. Typhimurium, up to 91%. Lb. rhamnosus 0900 inhibited E. coli by 75.9%, and Lb. casei 0919 decreased adherence of C. albicans by 49%. All pathogens activated the adherence of the mixture of probiotic bacteria.     

Purification and characterisation of a novel α‐L‐rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae

A novel α‐L‐rhamnosidase was isolated and purified from Aspergillus oryzae NL‐1. The enzyme was purified 13.2‐fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The K_M, kcat and kcat/K_M values were 5.2 mm , 1624 s^?1 and 312 s^?1 mm ^?1 using pNPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Modelling of the Enzymatic Hydrolysis of Potato (Solanum tuberosum) Using Response Surface Methodology

Starch hydrolysates containing glucose can be obtained from potato (Solanum tuberosum). Acid hydrolysis generates a solution that must be neutralized to be used for fermentative purposes while an enzymatic hydrolysis generates a glucose solution which can be used without further treatments. This work deals with the modelling of the enzymatic hydrolysis of dried potatoes with α‐amylase and glucoamylase with temperature, time, activity units/substrate ratio and α‐amylase activity fraction as operational variables and released glucose concentration as dependent variable. The model obtained showed that the optimal conditions were 70ºC, 60 min; 74 activity units/g substrate and 0.13 α‐amylase activity U/total U fraction. Using these conditions, a glucose concentration of 38.9 g/L was predicted which corresponded well with an experimental value of 38.0 ± 0.5 g/L. The model obtained can be used in the development of processes for the hydrolysis‐fermentation of potato for the production of food additives or ethanol.     

Phenotypic and molecular assessment of antimicrobial resistance in Lactobacillus paracasei strains of food origin

Antimicrobial resistance data in food-associated lactic acid bacteria (LAB) such as lactobacilli are mostly based on nonstandardized methodologies and/or have been obtained for only a limited number of strains. This susceptibility study included a diverse collection of 115 isolates mainly of food origin originally identified as Lactobacillus paracasei or Lactobacillus casei. Upon reidentification and removal of potential replicate isolates using repetitive DNA element PCR fingerprinting, 65 genotypically unique L. paracasei strains and the L. casei type strain were selected for broth microdilution and Etest assays using the LAB susceptibility test medium. In both methodologies, strains appeared uniformly susceptible to ampicillin and clindamycin but exhibited natural resistance to streptomycin and gentamicin. Three L. paracasei strains from cheese displayed acquired resistance to tetracycline (MIC > or = 32 microg/ml) and/or to erythromycin (MIC >16 microg/ml), which was linked to the presence of a tet(M) or tet(W) gene and/or an erm(B) gene, respectively. Partial sequencing revealed that the tet(M) genes found in two of these strains belonged to two tet(M) sequence homology groups previously found in enterococci. Collectively, phenotypic and genotypic data allowed us to propose tentative epidemiological cutoffs for L. paracasei and L. casei for differentiating susceptible strains from those strains harboring one or more acquired resistance factors.     

Effects of Mentha longifolia L. essential oil on viability and cellular ultrastructure of Lactobacillus casei during ripening of probiotic Feta cheese

The effects of Mentha longifolia L. essential oil during ripening and storage probiotic Feta cheese were studied, in relation to viability and cellular ultrastructure of Lactobacillus casei. The addition of the essential oil in the concentrations from 0.0 to 0.03% was trialled: the 0.03% treatment resulted in the highest viability of L. casei and the lowest pH value compared with other treatments (P < 0.05). Electron microscopy showed that essential oil caused no harm to L. casei. This study demonstrated the favourable effects of M. longifolia on optimal maintenance of L. casei at the end of cheese storage period.     

Light‐induced glycoalkaloid accumulation of potato tubers (Solanum tuberosum L)

The total glycoalkaloid concentration of non‐sprouted potato tubers, cvs Marfona, Fianna, Maris Piper, Kestrel and Golden Wonder, was measured following 15 days of continuous illumination (250 µmol m⁻² s⁻¹ photosynthetically active radiation) using high‐performance liquid chromatography. Comparisons were made of the influence of four sources of illumination: fluorescent tube‐type warm white, high‐pressure sodium, high‐pressure mercury types MB/U and MBFR/U. Irrespective of cultivar, glycoalkaloid concentrations of tubers placed in the dark remained relatively constant from day 0 to day 15 and did not exceed food safety values of 200 mg kg⁻¹ FW. A highly significant cultivar, light and cultivar × light (P < 0.01) interaction was recorded. Cultivar Kestrel was shown to be light‐sensitive with high rates of glycoalkaloid accumulation irrespective of light source, and cv Maris Piper was demonstrated to be light‐insensitive. In the majority of cultivars tested, maximal rates of glycoalkaloid accumulation were recorded following exposure of tubers to sodium or fluorescent light, and minimal rates of accumulation were recorded following exposure of tubers to mercury (MB/U and MBFR/U) illumination. In virtually all cases glycoalkaloid concentrations steadily increased with time during light exposure with no indication of cessation. In some instances the α‐chaconine/α‐solanine ratio decreased over 15 days of illumination (P < 0.05), indicating enhanced α‐solanine synthesis over that of α‐chaconine. The implications of these results in terms of consumer safety are discussed. © 1999 Society of Chemical Industry     

Is beer a source of prebiotics?

Beer contains low‐molecular‐weight β‐linked oligosaccharides that originate from the degradation of β‐glucan in the barley cell wall during malting and mashing. Over 90% of these oligosaccharides contain three or four glucosyl units. They remain intact through a static oral, gastric and small intestinal in vitro human digestive system model, indicating that they should be available to beneficial organisms known to be present in the human large intestine. Several intestine‐associated Lactobacillus strains were shown to be capable of growth on these β‐linked oligosaccharides, thereby leading us to tentatively propose that these compounds may represent prebiotics. Copyright © 2017 The Institute of Brewing & Distilling     

Effect of Infrared Blanching on Enzyme Activity and Retention of β‐Carotene and Vitamin C in Dried Mango

The objectives of this work were to evaluate infrared (IR) dry blanching in comparison with conventional water blanching prior to hot air drying of mango to inactivate polyphenol oxidase (PPO) and ascorbic acid oxidase (AAO) enzymes, and to study its effect on color change and retention of vitamin C and β‐carotene. Mango cylinders were blanched under similar temperature–time conditions either by IR heating or by immersion in a water bath during 2 min at 90 °C (high‐temperature‐short‐time—HTST) or for 10 min at 65 °C (low‐temperature‐long‐time—LTLT). After blanching mango was hot air dried at 70 °C. PPO was completely inactivated during the blanching treatments, but AAO had a moderate remaining activity after LTLT treatment (～30%) and a low remaining activity after HTST treatment (9% to 15%). A higher retention of vitamin C was observed in mango subjected to IR dry blanching, 88.3 ± 1.0% (HTST) and 69.2 ± 2.9% (LTLT), compared with water blanching, 61.4 ± 5.3% (HTST) and 50.7 ± 9.6% (LTLT). All‐trans‐β‐carotene retention was significantly higher in water blanched dried mango, 93.2 ± 5.2% (LTLT) and 91.4 ± 5.1% (HTST), compared with IR dry blanched, 73.6 ± 3.6% (LTLT) and 76.9 ± 2.9% (HTST). Increased levels of 13‐cis‐β‐carotene isomer were detected only in IR dry blanched mango, and the corresponding dried mango also had a slightly darker color. IR blanching of mango prior to drying can improve the retention of vitamin C, but not the retention of carotenoids, which showed to be more dependent on the temperature than the blanching process. A reduction of drying time was observed in LTLT IR‐blanching mango.     

A dark brown roast coffee blend is less effective at stimulating gastric acid secretion in healthy volunteers compared to a medium roast market blend

Coffee consumption sometimes is associated with symptoms of stomach discomfort. This work aimed to elucidate whether two coffee beverages, containing similar amounts of caffeine, but differing in their concentrations of ^βN‐alkanoyl‐5‐hydroxytryptamides (C5HTs), chlorogenic acids (CGAs), trigonelline, and N‐methylpyridinium (N‐MP) have different effects on gastric acid secretion in healthy volunteers. The intragastric pH after administration of bicarbonate with/without 200 mL of a coffee beverage prepared from a market blend or dark roast blend was analyzed in nine healthy volunteers. Coffee beverages were analyzed for their contents of C5HT, N‐MP, trigonelline, CGAs, and caffeine using HPLC‐DAD and HPLC‐MS/MS. Chemical analysis revealed higher concentrations of N‐MP for the dark brown blend (87 mg/L) compared to the market blend coffee (29 mg/L), whereas concentrations of C5HT (0.012 versus 0.343 mg/L), CGAs (323 versus 1126 mg/L), and trigonelline (119 versus 343 mg/L) were lower, and caffeine concentrations were similar (607 versus 674 mg/mL). Gastric acid secretion was less effectively stimulated after administration of the dark roast blend coffee compared to the market blend. Future studies are warranted to verify whether a high ratio of N‐MP to C5HT and CGAs is beneficial for reducing coffee‐associated gastric acid secretion.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

An application in Gouda cheese manufacture for a strain of Lactobacillus helveticus ND01

Gouda cheese was manufactured with Lactococcus lactis ssp. lactis IMAU60010, L. lactis ssp. cremoris IMAU40136 and L. helveticus ND01 isolated from the naturally fermented milk in China. Starter cultures added with L. helveticus ND01 produced Gouda cheese with dramatically more proteolysis than control cheeses. Compared with control cheese, experimental cheese with L. helveticus ND01 adjunct revealed dramatic increase in both Angiotensin I‐converting enzyme (ACE)‐inhibitory activity and γ‐aminobutyric acid content. The ACE‐inhibitory activity of Gouda cheeses with the addition of 1 × 10⁵ CFU/mL L. helveticus ND01 increases from 53.7 to 83.1% at 6 weeks of ripening.     

Assessment of changes in hop resins and polyphenols during long‐term storage

Changes in the content and composition of hop secondary metabolites during storage are reflected in beer quality and in the economics of beer production. A 12‐month storage experiment with T90 pellets of four hop varieties showed different dynamics of hop aging in relation to both storage conditions and hop variety. Negligible effects on the α‐ and β‐acids were detected during storage without air access at +2°C. Storage at +20°C resulted in a final loss of 20–25% α‐acids, but the content of β‐acids did not change significantly. Large decreases in α‐acids (64–88%) and in β‐acids (51–83%) were found in hops stored with access to air at +20°C. The rate of decline accelerated markedly after 6 months of storage. In terms of hop resin changes, Premiant and Sládek were the most and the least stable varieties, respectively. After 12 months, the content of the total polyphenols and flavonoids decreased by 30–40% and by 20–30%, respectively, irrespective of storage conditions. The rate of decline accelerated strongly after 6 months. The DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) antiradical potential decrease was significant only in hops stored under aerobic conditions. The depletion was 9–25% after 1 year; Saaz was the most stable variety. Copyright © 2012 The Institute of Brewing & Distilling     

Effect of lactulose on biotransformation of isoflavone glycosides to aglycones in soymilk by lactobacilli

ABSTRACT:  Lactobacillus acidophilus 4461, L. acidophilus 4962, L. casei 290, and L. casei 2607 were used to hydrolyze isoflavone glycosides (IG) to biologically active forms—isoflavone aglycones (IA)—in soymilk (SM) prepared from soy protein isolate (SPI) and soymilk supplemented with 0.5% (w/v) of lactulose (SML). L. acidophilus 4461 utilized the highest level of lactulose (3.01 mg/mL) and L. acidophilus 4962 utilized the least (0.86 mg/mL) at 24 h of incubation. The pH values decreased to 4.00 to 5.00 in SML, while they remained relatively high (6.15 to 6.36) in SM. Supplementation with lactulose significantly ( P < 0.05) enhanced the viable counts of all the 4 Lactobacillus strains. At the end of incubation, the viable counts of Lactobacillus ranged from 8.08 to 8.25 log CFU/mL in SML compared to 6.99 to 7.11 log CFU/mL in SM. Supplementation with lactulose increased the biotransformation of IG to IA after 6 h of incubation. The presence of lactulose in the medium enhanced the biotransformation level of IG to IA by Lactobacillus up to 21.9%. The hydrolysis level of malonyl genistin and acetyl genistin in SML was much higher than in SM by all the 4 probiotic organisms. The biotransformation of IG to IA occurred rapidly during the 1st 12 h of incubation in both SML and SM. Among the 4 Lactobacillus strains, L. acidophilus 4461 biotransformed the highest level (88.8%) of IG to IA in SML compared to 68.2% in SM after 24 h of incubation.     

Effect of cold shock on the enhancement of β‐amylase activity during malting and malt processability for a red sorghum intended for brewing use

The low β‐amylase activity of sorghum malt is a major concern when malts are intended for use in brewing. Several studies have shown that the germination temperature plays an important role in β‐amylase synthesis. In this study, the cold shock treatment was envisioned as a means of improving β‐amylase synthesis during red sorghum malting. The results show that, when a high‐frequency decrease in the germination temperature is used, the obtained malt exhibits a significantly increased β‐amylase activity. This study shows that this increase is not sufficient to consider cold shock as a means of improving β‐amylase activity for red sorghum brewing use, as the processabilty of the malts is unsatisfactory. Copyright © 2015 The Institute of Brewing & Distilling