Quantification of Lactulose and Epilactose in the Presence of Lactose in Milk using a dual HPLC analysis

The valuable lactose derivatives lactulose and epilactose can be derived from lactose either by the Lobry de Bruyn-Alberda van Ekenstein transformation during heat treatments or by enzymatic conversion using cellobiose 2-epimerases (EC 5.1.3.11). The chromatographic determination of lactose, lactulose, and epilactose in milk is challenging, due to the variable ratio of the three saccharides and their similar retention properties. In this work, a dual high-performance liquid chromatography (HPLC) analysis for the quantification of lactose, lactulose, and epilactose in milk samples was developed and validated. The samples originated from an enzymatic lactose conversion using the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Application of this enzyme led to the formation of high lactulose concentrations (28.0 g/L) in milk. The dual HPLC analysis utilized a combination of two chromatographic separation techniques, configured in two parallel systems. After precolumn derivatization, the samples were analyzed as follows: Method 1 determined the concentration of lactose and epilactose using a C₁₈ column with an ion-pair reagent as eluent, coupled with a UV detector. Method 2 determined the concentration of lactulose using a trimodal stationary phase (hydrophilic interaction, anion- and cation-exchange properties) with acetonitrile/ammonium formiate buffer as eluent, coupled with an evaporative light scattering detector. Both methods were validated in terms of linearity, precision and recovery. The revealing detection limits in the milk samples were 3.32 mg/L for lactose, 4.73 mg/L for epilactose and 139 mg/L for lactulose. The dual HPLC analysis presented allows accurate lactose, lactulose, and epilactose separation in complex food matrices such as milk.     

Detection and quantification of Escherichia coli and Pseudomonas aeruginosa in cow milk by near‐infrared spectroscopy

This work investigated the potential of NIR technology to be applied in the dairy industry for the detection of micro‐organisms. To this end, two types of cow milk samples were studied, one in which only bacterial biomass was considered and the other in which bacteria were cultured and grown in milk for 24 h. The study was carried out using two micro‐organisms Escherichia coli and Pseudomonas aeruginosa. Both types of samples with different counts of both micro‐organisms were analysed by a NIR analyser in the range 10 000–4000 cm^?1 based on transmittance measurements. Multivariate models indicated that a better discrimination between micro‐organisms was attained in those milk samples in which micro‐organisms have been grown.     

Lactulose formation catalysed by alkaline-substituted sepiolites in milk permeate

Alkaline-substituted (Na⁺, K⁺) sepiolites were used as catalysts for the formation of lactulose in milk permeate. Besides lactose and lactulose, other carbohydrates, such as galactose and epilactose, produced in side reactions, were determined. The effect of different washing cycles of sepiolite on the isomerisation of lactose and the exchange of cations with the permeate were also investigated. In general, the activity of the sodium sepiolite was higher than that of potassium form. Twenty per cent of lactulose formation (≈1000 mg/100 ml), with respect to the initial lactose, was obtained after 150 min of reaction, using sodium sepiolite washed during 10 cycles. Under these conditions, 25% of lactose degradation was detected, whereas small amounts of epilactose and galactose were formed. The exchange of Na⁺ between sepiolite and permeate decreased considerably with the number of washing cycles. The present work shows an appropriate method for obtaining lactulose in milk permeate with acceptable yields and without complicated purification steps.     

Phenolic acids from malt are efficient acetylcholinesterase and butyrylcholinesterase inhibitors

Anticholinesterase activities of mashes produced using wheat (‘Wheat Pale’) or barley malts (‘Pilsner’, ‘Pale Ale’, ‘Munich Light’, ‘Carahell’ or ‘Carared’) were studied by spectrophotometric method. The highest inhibition of acetylcholinesterase and butyrylcholinesterase was observed at 52 °C and/or 64 °C, followed by a decrease or stabilization of the activity at 72 °C. Changes in the total phenolics content in the test mashes were correlated with changes in the acetylcholinesterase and/or butyrylcholinesterase activities. Phenolic acids were singled out from phenolic compounds for more detailed studies owing to their simplicity and structural similarity to well‐known cholinesterase inhibitors. The main phenolic acids in the test malts were ferulic, gallic, p‐coumaric and vanillic acids followed by chlorogenic, caffeic, syringic, p‐OH‐benzoic, sinapic and protocatechuic acids. The anticholinesterase activities of the phenolic acids were studied using model standard solutions at concentrations similar to the maximal content of these compounds in the test mashes. Among the phenolic acids, p‐coumaric acid had the largest share in the anticholinesterase activity, even though it was present in the test mashes at a significantly lower concentration (~0.38 mm L^?1) than ferulic acid (~1.00 mm L^?1). Sinapic acid and p‐OH‐benzoic acid (0.03 and 0.01 mm L^?1, respectively) were equally efficient inhibitors as ferulic acid at ~1.00 mm L^?1. This preliminary study should be extended to other phenolic compounds from malt (wort) in the near future. Copyright © 2012 The Institute of Brewing & Distilling     

Encapsulation in an alginate–goats’ milk–inulin matrix improves survival of probiotic Bifidobacterium in simulated gastrointestinal conditions and goats’ milk yoghurt

In this work, a new encapsulating matrix, alginate–goats’ milk–inulin, was used to encapsulate Bifidobacterium animalis subsp. lactis BB‐12. The addition of inulin resulted in capsules with a compact structure, and a higher probiotic cell count under simulated gastrointestinal conditions and in probiotic goats’ milk yoghurt during refrigerated storage. Encapsulation of the probiotic bacteria led to slower post‐acidification yoghurts. The results of this study showed that the alginate–goats’ milk–inulin matrix has potential to be used as a new encapsulation material to encapsulate probiotics for use in goats’ milk‐based probiotic fermented dairy products, avoiding the cross‐contamination caused by using capsules based on cows’ milk.     

Chemical analysis and acetylcholinesterase inhibitory effect of anthocyanin-rich red leaf tea (cv. Sunrouge)

BACKGROUND: The purpose of this study was to evaluate the effects of leaf order or crop season on anthocyanins and other chemicals in the anthocyanin‐rich tea cultivar ‘Sunrouge’ (Camellia sinensis x C. taliensis) by using high‐performance liquid chromatography, and to study the effect of ‘Sunrouge’ extract on acetylcholinesterase (AChE) activity in human neuroblastoma SK‐N‐SH cells. RESULTS: The total anthocyanin content was higher in the third (3.09 mg g^?1) than in the second (2.24 mg g^?1) or first crop season (1.79 mg g^?1). The amount of anthocyanins contained in the stem was high (1.61 mg g^?1). In the third crop season, the concentrations of delphinidin‐3‐O‐β‐D ‐(6‐(E)‐p‐coumaroyl)galactopyranoside (DCGa), cyanidin‐3‐O‐β‐D ‐(6‐(E)‐p‐coumaroyl)galactopyranoside, delphinidin‐3‐O‐β‐D ‐galactopyranoside, delphinidin‐3‐O‐β‐D ‐(6‐O‐(Z)‐p‐coumaroyl)galactopyranoside, cyanidin‐3‐O‐β‐D ‐galactoside, and delphinidin‐3‐O‐β‐D ‐glucoside were 1.57 mg g^?1, 0.52 mg g^?1, 0.40 mg g^?1, 0.22 mg g^?1, 0.14 mg g^?1, and 0.11 mg g^?1, respectively. DCGa accounted for about 50% of the anthocyanins present. The suppressive effect of ‘Sunrouge’ water extract on AChE activity in human neuroblastoma SK‐N‐SH cells was the strongest among the three tea cultivars (‘Sunrouge’, ‘Yabukita’ and ‘Benifuuki’). CONCLUSION: These results suggested that ‘Sunrouge’ might protect humans from humans from AChE‐related diseases by suppressing AChE activity. To obtain sufficient amounts of anthocyanins, catechins and/or caffeine for a functional food material, ‘Sunrouge’ from the third crop season should be used. Copyright © 2012 Society of Chemical Industry     

Effects of heating temperatures and addition of reconstituted milk on the heat indicators in milk

The objective of the present study was to evaluate the effect of heating temperatures and reconstituted milk on heat treatment indicators in milk by comparing the heat damage between raw milk and raw milk plus reconstituted milk (composite milk). The contents of lactulose, furosine, beta-lactoglobulin, and lactoperoxidase were determined after the heat indicators were heated to 65 to 115 °C for 15 s both in raw milk and composite milk. In the raw milk, the lactulose and furosine contents increased with increased heating temperature, while the beta-lactoglobulin content and lactoperoxidase activity decreased. The lactulose and furosine contents were increased after the addition of reconstituted milk. The reconstituted milk also significantly (P < 0.05) reduced the concentration of beta-lactoglobulin in the milk. Both heat treatment and an addition of reconstituted milk decreased the lactoperoxidase activity significantly (P < 0.05), and the lactoperoxidase activity was undetectable at 85 °C. The ratios of lactulose to furosine in pasteurized milk were higher than that in composite pasteurized milk. It is concluded that lactulose, furosine, and beta-lactoglobulin are suitable indicators of high heat pasteurization or raw milk, while lactoperoxidase may be used in monitoring mild heat pasteurization. Practical Application: Adequate heat treatment is necessary to destroy the microbes in raw milk. However, excessive heat treatment can result in inactivation of active compounds or loss of nutrients. The present study showed that the concentrations of lactulose, furosine, beta-lactoglobulin, and the activity of lactoperoxidase are sensitive to processing temperature and can serve as indicators of milk pasteurization.     

高效液相色谱法测定液态乳中乳果糖方法的优化

为了对液态乳中乳果糖含量进行简便准确地定量,改进高效液相色谱法测定乳果糖含量的方法.采用化学稳定的改性酰胺基色谱柱进行分离,并以乙腈-水流动相梯度洗脱40 min,结果表明,样品进样体积为3 μL时乳糖与乳果糖的分离度为1.5,满足色谱分离检测.优化后的方法经验证,乳果糖在50～2500 mg/kg范围内线性关系良好,...     

Occurrence of endosulphan residues in dairy milk in plains of Uttarakhand,India: Short communication

Endosulphan residues were determined in milk samples collected from various locations of plains of Uttarakhand covering Tarai and Kumaon regions. Residues were extracted from milk by liquid–liquid partition followed by clean‐up by alumina column chromatography. High‐performance liquid chromatography (HPLC) was used for the detection and quantification of residues. Of the total 170 milk samples collected from different species, 1.17% samples showed residues of endosulphan‐alpha; 2.35% endosulphan β and 4.7% milk samples showed endosulphan‐sulphate residues with mean residual concentrations of 0.244, 0.566 and 0.265 μg/mL, respectively. About 6.47% of milk samples showed endosulphan residues above the maximum residue limit (MRL) of 0.1 mg/kg.     

Maillard Reaction in Microwave Cooking: Comparison of Early Maillard Products in Conventionally and Microwave-Heated Milk

The impact of microwave cooking on the formation of early Maillard products was investigated and compared with the effect of conventional cooking, using milk as a test system. Experiments were carried out at controlled temperatures of 80°C and 90°C, respectively, at holding times up to 420min. Furosine, hydroxymethylfurfural (HMF) and lactulose, which are all established indicators to estimate heat damage, were determined. The concentrations of all the heating indicators increased with increasing heating time. For example in the 90°C test series the furosine values rose from 34mglitre⁻¹ (0·5h) to 94mglitre⁻¹ (2h holding time) in the milk heated by microwaves and from 35mglitre⁻¹ (0·5h) to 96mg litre⁻¹ (2h) in the conventionally heated milk. None of the reaction products showed significant differences as between the microwave heating and conventional cooking methods.     

Distinguishing pasteurized, UHT and sterilized milks by their lactulose content

Commercial pasteurized, UHT and sterilized milk samples, and laboratory autoclaved milks, were analysed for lactulose by an enzymatic method. No lactulose was detected in the pasteurized milk. On the basis of the measured lactulose concentrations, the UHT and sterilized milk samples fell into five groups according to the type of process that they had received.
The lactulose concentrations in the laboratory autoclaved milks rose on storage, but this was not observed in the other milks.
It appears that pasteurized, UHT and sterilized milks can be differentiated by their lactulose content.     

Consumption of raw brown onions variably modulate plasma lipid profile and lipoprotein oxidation in pigs fed a high‐fat diet

This study was undertaken to determine the effects of two commercially available brown onion varieties, ‘Cavalier’ and ‘Destiny’, supplemented at two different levels, on blood lipid and oxidative status using the pig as a model. Twenty‐five female cross‐bred pigs were allocated to one of five dietary treatments that consisted of a high‐fat control diet with no onion added, a low onion dose of 10 g onion MJ^?1 DE and a high dose of 25 g onion MJ^?1 DE for each variety of onion. Supplementation with ‘Destiny’ onion resulted in a 21% (p < 0.05) reduction in the averaged fasted and postprandial plasma triacylglyceride (TG) measurements taken over the six‐week period in comparison with the control pigs. The average fasting and postprandial plasma cholesterol concentrations were significantly reduced by 5.5 and 12.4% in pigs that consumed the low and high dose of ‘Destiny’ onion, respectively (p < 0.010), while ‘Cavalier’ was only effective at lowering cholesterol levels by 10% at the lower dose of supplementation. Inhibition in the rate of serum lipoprotein oxidation, measured as lag time, was increased by 23% (p < 0.05) in plasma obtained from pigs that consumed ‘Cavalier’ compared with the control and ‘Destiny’ onion diets. These data indicate that onion consumption level may provide a dietary means of manipulating some of the risk indices associated with coronary heart disease, but the responses varied with type and dose of onion. Copyright © 2004 Society of Chemical Industry     

Evaluation of Pseudomonas spp. through O2 and CO2 headspace analysis

This article proposes an approach to determine the level of Pseudomonas spp. in milk, based on the evaluation of the content of oxygen and carbon dioxide produced in the headspace of sealed vials; the research was divided into two phases: model building and preliminary validation. Three different strains of Pseudomonas spp., Ps. putida (wild strain) and Ps. fluorescens (wild and collection isolates), were used as targets. Data of CO₂ and O₂ were modelled through a modified positive (CO₂) or a negative Gompertz equation (O₂) to estimate the Minimum Detection Time (MDT), defined as the time to attain 3% of CO₂ (MDT₁) or a decrease in the content of O₂ by 3% (MDT₂). Then, MDT₁ and MDT₂ were submitted to a linear regression procedure, using cell concentration as independent variable; the correlations ‘MDT₁/cell concentration’ and ‘MDT₂/cell concentration’ showed high determination coefficients (>0.983). Moreover, the regression procedure pointed out that both MDT₁ and MDT₂ decreased by ca. 3 h for an increase in cell count of 1 log cfu mL^?1. Preliminary validation in milk pointed out that the error associated with the regression line ‘MDT₂/cell concentration’ was below 5%.     

Evaluation of colouring ability of main European elderberry (Sambucus nigra L.) varieties as potential resources of natural food colourants

Profiling and quantitative analysis of anthocyanins in five elderberry (Sambucus nigra L.) varieties, namely ‘Haschberg’, ‘Samocco’, ‘Samyl’, ‘Samident’ and ‘Sampo’, were performed in six different maturity stages from two consecutive years (2012 and 2013) and from two growing areas in Hungary. Cyanidin‐3‐O‐sambubioside‐5‐O‐glucoside, cyanidin‐3‐O‐sambubioside and cyanidin‐3‐O‐glucoside were found and identified by HPLC‐Q/TOF‐MS as major anthocyanins and were quantified by HPLC‐UV/Vis. In optimum maturity stage, ‘Samocco’ showed the highest anthocyanin content with an average of 1237 mg per 100 g dry weight in both growing areas and vintages. The dominant anthocyanin component of Samocco variety was cyanidin‐3‐O‐sambubioside, which is according to literature more stable against technology processing than cyanidin‐3‐O‐glucoside found in the other four investigated elderberry varieties in the highest concentration. ‘Samocco’, if grown under the climatic conditions of the Carpathian basin, might be a promising alternative variety for growing as raw material for natural food colourant processing industry.     

Anti-oxidant content of different coloured sweet peppers, white, green, yellow, orange and red (Capsicum annuum L.)

Five different coloured sweet peppers (Capsicum annuum cv. Signal), white, green, yellow, orange and red were analysed for total carotenoids, α‐tocopherol, sugars (glucose, fructose and sucrose), organic acids (citric and ascorbic acids) and anti‐oxidant properties. The mature fruits, ‘Signal Red’, ‘Signal Orange’ and ‘Signal Yellow’ contained higher carotenoids, α‐tocopherol, sugars and organic acids than the immature fruits, ‘Signal Green’ and ‘Signal White’. Among the mature fruits, ‘Signal Red’ was the highest in total carotenoids [9.15 mg (100 g)^?1 of fresh weight] while ‘Signal Orange’ was the highest in α‐tocopherol [5.40 mg (100 g)^?1 of fresh weight]. ‘Signal Red’ and ‘Signal Orange’ contained the most sugars and organic acids. The suppression of 2,2′‐azobis (2,4‐dimethylvaleronitrile) (AMVN)‐induced oxidation of methyl linoleate by the acetone extracts from the coloured sweet peppers resulted as follows: ‘Signal Red’ > ‘Signal Orange’ ≈ ‘Signal Yellow’ > ‘Signal Green’ ≈ ‘Signal White’. The order of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities of the acetone extracts was similar to that of suppression of methyl linoleate oxidation.     

Effect of the protein, citrate and phosphate content of milk on formation of lactulose during heat treatment

Milk ultrafiltrate and milks of varying protein, citrate and phosphate concentrations were heated in sealed containers. Protein was found not to be involved in the mechanism of formation of lactulose, but increasing the protein content of milk reduced the concentration of lactulose after heating. This was considered to be due to increased condensation of lactose and lactulose with amino groups of the protein. Less lactulose was formed in milk ultrafiltrate than in skimmed milk accorded the same heat treatment, which was attributed to the buffering capacity of the milk protein in skimmed milk. Activation energies for lactulose formation in skimmed milk and in ultrafiltrate were 128 and 131 kJ/mol respectively. Citrate and phosphate catalysed the formation of lactulose. It is proposed that the formation of free lactulose in heated milk and ultrafiltrate proceeds exclusively by the Lobry de Bruyn-Alberda van Ekenstein transformation with the naturally occurring phosphate and citrate acting as base catalysts.     

Formation and dispersal of biofilms in dairy substrates

The formation and dispersion of biofilms in the dairy industry is a problem because it increases cross‐contamination, affecting the shelf life of the products and their safety. The objective of this study was to evaluate the influence of different dairy substrates (cows’ milk and whey protein) on the formation and dispersion of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus in two biofilm systems (mono‐species and multi‐species) on stainless steel at 25 °C. The dominant behaviour of E. faecalis occurred in most of the tests on mono‐species and multi‐species biofilms. A greater dispersion of biofilm cells was observed in skimmed milk.     

Evaluation of the prebiotic potential of five kiwifruit cultivars after simulated gastrointestinal digestion and fermentation with human faecal bacteria

We studied the in vitro prebiotic potential of five different cultivars of kiwifruit including the green‐fleshed ‘Hayward’ and ‘Zesh004’ and the gold‐fleshed ‘Hort16A’, ‘Zesy002’ and ‘Zesy003’. The kiwifruit (25 g fresh weight equivalent) were subjected to simulated gastrointestinal digestion before fermentation for 16 h with faecal microbiota from ten individual donors. Microbial metabolites including lactate were quantified while changes in microbiome composition were determined by 16S rRNA sequencing. Lactate concentrations were highest with ‘Hayward’ (P = 0.01) and correlated with the amount of the kiwifruit fibre and polyphenols, chlorogenic acid and cryptochlorogenic acid. All the kiwifruit behaved similar to inulin in increasing the relative abundance of Bifidobacterium (P < 0.001), but unlike inulin, significantly (P < 0.001) increased the abundances of Ruminococcaceae and decreased Bacteroides. In comparison with inulin, the green‐fleshed kiwifruit selectively increased Lachnospira (P = 0.008) while the gold‐fleshed kiwi fruit increased Akkermansia (P < 0.001). These data suggest that the fibre and polyphenol content of the kiwifruit play a role in modulating gut microbial metabolism. Further clinical studies with these kiwifruit cultivars are required to confirm the potential prebiotic benefits that may be achieved by normal dietary intervention.     

Lactose content of modified enzyme-treated ‘dadih’

The preparation of ‘dadih’, a sweet ‘gel-like’ fresh milk product which is a popular dessert in the northern part of Peninsular Malaysia is described. The lactose content of modified ‘dadih’ and enzyme-treated ‘dadih’ was monitored throughout 7 days, a period over which ‘dadih’ is normally stored and kept at 4°C. Modified ‘dadih’, made from fresh milk with 4.49% lactose, yields 3.63% lactose upon formulation, a value still high for lactose-intolerant consumers. A suitable volume of commercially-prepared enzyme, Lactozym 3000L to hydrolyse lactose in ‘dadih’ was found to be 1 U ml⁻¹, yielding >70% hydrolysis after a 48 h incubation period at 4°C. The colour of enzyme-treated ‘dadih’ was significantly different (p<0.05) from the untreated samples. Other characteristics, such as texture, aroma and overall acceptability, showed no significant differences (p>0.05). ©