Development of a quantitative immuno-affinity test column assay for on-site screening of clindamycin residues in milk

A sensitive and specific monoclonal antibody against clindamycin (CLIN) was produced and used to develop an immuno-affinity test column (IATC) assay for on-site screening of CLIN residues in milk. The qualitative limit of detection of the IATC assay, estimated as 1.0 μg L⁻¹ by visual detection, was sufficient to measure maximum residue levels for lincosamide antibiotics in milk. The quantitative IATC assay resulted in a lower detection limit (0.11 μg L⁻¹) by evaluating the colour intensity of the test layers, which was approximately 9 times greater sensitivity than visual detection. During the spike and recovery test, the average recoveries ranged from 76% to 114% at different spiked levels, and the intra-/interday coefficients of variation were in the range 9.6–16.7%. The detection time was shortened to 20 min, whereas the sensitivity was comparable with a traditional ELISA. The IATC assay shows promise for on-site screening of CLIN residues in milk.     

Performance of whey protein hydrolysate–maltodextrin conjugates as emulsifiers in model infant formula emulsions

Model infant formula emulsions containing 15.5, 35.0 and 70.0 g L⁻¹ protein, soybean oil and maltodextrin (MD), respectively, were prepared. Emulsions were stabilised by whey protein hydrolysate (WPH) + CITREM (9 g L⁻¹), WPH + lecithin (9 g L⁻¹) or WPH conjugated with MD (WPH–MD). All emulsions had mono-modal oil droplet size distributions post-homogenisation with mean oil droplet diameters (D_4,3) of <1.0 μm. No changes in the D_4,3 were observed after heat treatment (95 °C, 15 min) of the emulsions. Accelerated storage (40 °C, 10 d) of unheated emulsions resulted in an increase in D_4,3 for CITREM (2.86 μm) and lecithin (5.36 μm) containing emulsions. Heated emulsions displayed better stability to accelerated storage with no increase in D_4,3 for CITREM and an increase in D_4,3 for lecithin (2.71 μm) containing emulsions. No increase in D_4,3 over storage was observed for unheated or heated WPH–MD emulsion, indicating its superior stability.     

Galacto-oligosaccharide synthesis using chemically modified β-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin

The goal of this study was to establish an efficient immobilisation protocol for β-galactosidase from Aspergillus oryzae onto the polystyrenic macroporous resin Purolite^® A-109 for better utilisation of its transglactosylation activity and application in galacto-oligosaccharide (GOS) synthesis. This was achieved by improving simple ionic adsorption by carboxyl group activation on the enzyme surface with carbodiimide, enabling covalent immobilisation. This yielded significantly increased operational stability, assayed as GOS synthesis, in a batch reactor, and even more prominently, in a fluidised bed reactor (73% activity retained after 10 cycles). The immobilised enzyme showed two very beneficial advantages over the free enzyme for future applications: higher affinity towards catalysing transgalactosylation than towards hydrolysis and shift of pH optimum towards more acidic conditions. GOS synthesis performed under the optimum conditions obtained (400 g L⁻¹ lactose, pH 4.5, 50 °C) yielded 87 g L⁻¹ and 100 g L⁻¹ for batch and fluidised bed reactors, respectively.     

Determination of menaquinone production by Lactococcus spp. and propionibacteria in cheese

Two genera of lactic acid bacteria are principally known to produce menaquinones in cheese: Lactococcus spp. in semi-hard cheese such as Vacherin Fribourgeois and Raclette, and propionibacteria in Swiss-type cheese such as Emmental. Menaquinone (MK) content of several cheese loaves was analysed to determine the impact of sampling site, ripening time and cultures used during cheese making. With Lactococcus spp., in Vacherin and in Raclette, the principal menaquinone was MK-9 (median = 149 μg kg⁻¹ and median = 167 μg kg⁻¹) followed by MK-8 (median = 70 μg kg⁻¹ and median = 66 μg kg⁻¹). In Emmental cheese, the principal menaquinone was MK-4 (median = 48 μg kg⁻¹) in young cheese and MK-9(4H) (median = 468 μg kg⁻¹) in cheese older than 90 days. The only difference in sampling site was found in Raclette for MK-9. In Vacherin, MK-8, MK-9 and total contents of menaquinones were significantly different according to the strain of Lactococcus lactis ssp. cremoris used.     

Selenium supplementation of diets of dairy cows to produce Se-enriched cheese

Selenium (Se) deficiency in people and animals is a nutritional problem in many regions of the world. Ten mid-lactation Estonian Red dairy cows were supplemented for 64 days with inorganic Se [0.39 mg kg⁻¹ in total mixed ration (TMR)] followed by a 57-day period of supplementation with organic and inorganic Se (0.44 mg kg⁻¹ in TMR), according to EU directives on maximum allowed amounts. Feeding organic Se increased Se content in blood (from 186.5 to 287.9 μg kg⁻¹), milk (from 17.1 to 51.8 μg kg⁻¹) and Edam-type cheese made there from (from 146 to 361 μg kg⁻¹). Se content in milk after supplementation was high enough to produce a cheese enabling the nutrition claim “high in Se” and related health claims. The concentration of the main primary oxidation products of linoleic acid (oxylipins) was low and leukotoxin diols were found in trace amounts; the oxidative stability of cheeses was high.     

Determination of aflatoxins in eggs,milk, meat and meat products using HPLC fluorescent and UV detectors

Raw and pasteurised sheep’s, cow’s and goat’s milk, eggs, and beef samples from different local markets in Jordan were collected during a period of 5 months (January through May 2007) and examined for aflatoxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG2), M1(AFM1) and M2(AFM2). The samples were analysed with high performance liquid chromatography (HPLC) using UV and Fluorescent detectors. The analysed samples of milk collected in January were found to contain 0.56 μg L⁻¹ AFM1 and 0.1 μg L⁻¹ AFM2 whilst, the concentration of AFM1 and AFM2 was < 0.05 μg L⁻¹ for milk samples collected between March and May. The AFB1, AFB2, AFG1 and AFG2 contents in the analysed food products ranged from 1.10 to 8.32 μg L⁻¹ and 0.15 to 6.36 μg L⁻¹ in imported and fresh meat samples collected during March, respectively. The mean recovery for the HPLC method was 92% to 109% and the quantification levels were 50 ng L⁻¹ for AFM1 and AFM2. The AFM1 was found in 10% of the tested samples with concentrations between 0.08 and 1.1 μg kg⁻¹ and AFM2 was only found in 1.82% of the tested samples with a level of 0.1 μg kg⁻¹. The AFM1 levels in the examined foods were higher than the maximum level of AFM1 in liquid milk set by the European Community and Codex Alimentarius of 50 ng L⁻¹.     

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.     

Effect of chestnut tannin extract (Castanea sativa Miller) on the proliferation of Cladosporium cladosporioides on sheep cheese rind during the ripening

Strains belonging to the genus Cladosporium can cause black spots on the surface of sheep cheese, making it impossible to sell. Two water solutions of chestnut tannin extract (i) 200 g L⁻¹ (CHE200) and (ii) 400 g L⁻¹ (CHE400), and the chestnut tannin extract powder (CHEP) were tested in a cheese making trial in which 60 cheese units were allotted to 5 experimental groups (each of 12 cheeses: C1, control 1 without any treatment; C2, control 2 treated with a silver ion solution; and cheeses LCHE200, LCHE400, and LCHEP, treated with CHE200, CHE400, and CHEP, respectively). The cheeses were ripened in a room polluted with Cladosporium cladosporioides with the aim to create conditions for the proliferation of this fungus on the cheeses. The results indicated that chestnut tannin extract at a concentration of 200 g L⁻¹ is capable of completely inhibiting C. cladosporioides proliferation, avoiding spoilage of the sheep cheese.     

Physicochemical and microbiological description of Caxiri – a cassava and corn alcoholic beverage

Caxiri is a fermented alcoholic beverage made from cassava, corn and sweet potatoes by indigenous people in Brazil. Saccharomyces cerevisiae Rhodotorula mucilaginosa, Lactobacillus fermentum, Bacillus subtilis and L. helveticus were the main microbial species detected. Maltose was the main carbohydrate found (19.12 g L^?1), and lactic acid (15.09 g L^?1) and ethanol (92.16 g L^?1) were also found in high concentrations. Gas chromatography‐flame ionisation detector was used to identify thirteen volatile compounds. Among these volatiles, the higher concentrations were decanoic acid (123.04 μg L^?1) for the acids, diethyl malate (88.32 μg L^?1) for the esters, furfural (109.31 μg L^?1) for the aldehydes, 2‐phenylethanol (1022.76 μg L^?1) for the alcohols and 1,1‐diethoxyethane (226.24 μg L^?1) for the others. This study contributes to increasing knowledge of the microbiota present in the alcoholic fermentation produced from cassava, corn and sweet potatoes.     

Effect of fructooligosaccharides and galactooligosaccharides on the folate production of some folate-producing bacteria in media cultures or milk

The present work investigated the ability of Bifidobacterium catenulatum, Bifidobacterium adolescentis, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus to produce folate in milk and complex media. Moreover, the effect of two prebiotics, fructooligosaccharides and galactooligosaccharides, on folate biosynthesis was also evaluated. Levels of the predominant folate forms, i.e., tetrahydrofolate and 5-methyltetrahydrofolate, were determined using high performance liquid chromatography after 0, 6, 10 and 24 h incubation. B. catenulatum (28.82 ± 2.02 μg 100 mL⁻¹) and S. thermophilus (19.03 ± 1.95 μg 100 mL⁻¹) produced the highest level of folate in complex media and milk, respectively. In most cases, the bacteria tested reached the maximum folate levels within 6 h and 10 h of incubation. The inclusion of prebiotics in the culture medium did not stimulate the synthesis of folate by any of the five bacteria studied, although it increased the rate of bacterial growth.     

Immunomodulatory activities of Lactobacillus rhamnosus ZDY114 and donkey milk in BALB/c mice

The immunomodulatory activity of Lactobacillus rhamnosus ZDY114 and donkey milk in BALB/c mice was evaluated by assessing the splenic lymphocyte transformation, haemolytic complement activity, carbon clearance ability and natural killer cell activity. Results showed donkey milk (5 g kg⁻¹) in combination with L. rhamnosus ZDY114 (5 × 10⁷ cfu mL⁻¹) exhibited a significant increase in splenic lymphocyte proliferation, carbon granule engulfing ability and natural killer cell activity when compared with donkey milk or L. rhamnosus ZDY114 alone (p < 0.05). An elevated response in serum haemolytic activity was only observed when compared with L. rhamnosus ZDY114. In conclusion, donkey milk (5 g kg⁻¹) in combination with L. rhamnosus ZDY114 (5 × 10⁷ cfu mL⁻¹) was able to enhance specific immune functions.     

Development of a HPLC method combined with ultraviolet/diode array detection for determination of monosodium glutamate in various food samples

An effective, simple and rapid analytical method using HPLC was developed for the analysis of monosodium glutamate (MSG) in various food samples obtained from local market in Turkey. The determination of MSG was performed by its derivatisation with o-phthaldialdehyde (OPA). A high-performance liquid chromatography-ultraviolet/diode array detection method was performed by using C₁₈ (150 mm × 4.6 mm, 2.7 μm) column with the mobile phase consisting of 10 mm phosphate buffer solution (pH = 5.90) and methanol (75:25, v/v). The applied method was optimised and the validated. The method was linear from 1 to 50 μg mL⁻¹ of MSG. The correlation coefficient value of the developed method was obtained as R² = 0.9999. The limit of detection and limit of quantification limits were 0.015 and 0.050 μg mL⁻¹, respectively. MSG contents of the food samples range from 0.09 g kg⁻¹ to 120.80 g kg⁻¹. The validated method was successfully applied for the analysis of MSG in several food samples.     

Evaluation of various Bacillus coagulans isolates for the production of high purity L-lactic acid using defatted rice bran hydrolysates

Defatted rice bran (DRB) constitutes an abundant by-product stream, generated during rice milling and subsequent bran oil extraction. Enzymatic hydrolysis of starch and protein content in DRB was optimised in terms of solid loading. Among the four solid loadings evaluated (10%, 15%, 20% and 25%), the hydrolysate derived from 20% solids resulted in the highest concentration of glucose (82.3 g L⁻¹) and free amino nitrogen (234.8 mg L⁻¹). The fermentability of the hydrolysate was evaluated via screening of sixteen isolates. All the strains were able to grow and produce high purity L-lactic acid, utilising the DRB as sole carbon and nutrient source. Among the studied strains, the Bacillus coagulans A107 isolate presented the most promising results in terms of final lactic acid concentration (75.9 g L⁻¹), yield (0.90 g g⁻¹) and productivity (2.7 g L⁻¹ h⁻¹). The results of this study indicate that DRB could be employed as an inexpensive, alternative substrate for L-lactic acid production.     

Polyphenolic composition,antioxidant and antiproliferative effects of wild and cultivated blackberries (Rubus fruticosus L.) pomace

The content of total polyphenolics, antioxidative capacity and antiproliferative activity were tested in wild and cultivated blackberry pomace. Wild blackberry pomace extract Tw2 showed the highest following contents: total polyphenolics (50.16 mg GAE g⁻¹ dw), flavonoids (7.73 mg Qc g⁻¹ dw), flavonols (6.63 mg Qc g⁻¹ dw) and total monomeric anthocyanins (13.40 mg Cy g⁻¹). Tw2 extract significantly inhibited free radicals: IC₅₀^DPPH = 127.76 μg mL⁻¹, IC₅₀^ABTS = 26.53 μg mL⁻¹ and IC₅₀ ^{˙ OH} = 168.62 μg mL⁻¹, and the growth of breast adenocarcinoma IC₅₀^MCF7 = 306.68 μg mL⁻¹ and cervix epitheloid carcinoma cell lines IC₅₀^HeLa = 315.49 μg mL⁻¹. Wild blackberry varieties had higher extraction yields, higher total polyphenolic contents and possessed stronger biological effects compared to cultivated blackberries (P < 0.05). All blackberry extracts showed high biological potential that could be attributed to high total polyphenols and flavonoids content and could be utilised as value-added functional food.     

Determination of phospholipid concentrations in breast milk and serum using a high performance liquid chromatography–mass spectrometry–multiple reaction monitoring method

Phospholipids (PLs), including sphingomyelin, play important roles in cell membrane integrity, neural and brain development, and inflammatory responses. They are found in tissues and biological fluids, including human breast milk. In this cross-sectional study using high performance liquid chromatography–mass spectrometry, the average total PL concentrations in Malaysian mothers' colostrum and transitional milk were determined as 331 and 266 mg L⁻¹, respectively. In mature milk, the average total PL concentrations were 170, 210 and 220 mg L⁻¹ at 2, 6 and 12 months, respectively, with a strong correlation between the total PL concentration and the fat concentration (p < 0.001). The dominant PL class in mature milk was sphingomyelin (36–38%), followed by phosphatidylethanolamine (27–37%). The average maternal serum PL concentrations were higher in the third trimester (2089 mg L⁻¹) than in the second (1667 mg L⁻¹), with phosphatidylcholine predominant at 66% and with sphingomyelin at 22–24% of total PLs.     

Pilot-scale production of hydrolysates with altered bio-functionalities based on thermally-denatured whey protein isolate

Whey protein isolate (WPI) solutions (100 g L⁻¹ protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase^® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC₅₀) in solution. The lowest ACE inhibition concentration (IC₅₀ = 0.23 g L⁻¹ protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.     

Semiochemical modulation of host preference of Callosobruchus maculatus on legume seeds

Host preference of Callosobruchus maculatus (F.) on seeds of three legume cultivars, Ife-brown and black-eyed cowpeas [Vigna unguiculata L. (Walp)], and soybean (Glycine max L.), was investigated. Mated female C. maculatus showed high (90–95%) attraction to the three legume cultivars in Y-tube bioassays. However, the weevils discriminated among the cultivars in four-choice tests and showed greater attraction to Ife-brown cowpea (50%) than to soybean (30%) and black-eyed cowpea (15%). Coupled gas chromatography-electroantennography (GC-EAD) and GC–MS analyses of the headspace volatile organic compounds (VOCs) emitted by the legume seeds identified 2-ethyl hexanol as the principal EAD active component. Emission of 2-ethyl hexanol was two-fold greater in Ife-brown cowpea (∼0.54 μg g⁻¹ seeds) compared with black-eyed cowpea (∼0.23 μg g⁻¹ seeds) and soybean (∼0.21 μg g⁻¹ seeds). Synthetic 2-ethyl hexanol attracted 68% of female C. maculatus at 0.01 μg dose in Y-tube bioassays. These results demonstrated that host preference in C. maculatus is odor-mediated, and identified 2-ethyl hexanol as a potential attractant for C. maculatus.     

Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers

Enterococcus spp. contamination was screened from a Minas Frescal cheese processing line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was evaluated and the effect of sanitization procedures in the control of these biofilms was investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle, floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7, 13, 27, 41, and 47 °C) using response surface methodology. The model showed that E. faecium biofilms were formed from 1 to 8 days at 12–47 °C, while E. faecalis biofilms were formed from 1 to 8 days at 10–43 °C. None of the sanitizers (sodium hypochlorite 100 mg L⁻¹, peracetic acid 300 mg L⁻¹, and chlorhexidine digluconate 400 mg L⁻¹) was able to completely eliminate the biofilms.     

Changes in phenolics, γ-aminobutyric acid content and antioxidant,antihypertensive and hypoglycaemic properties during ale white sorghum (Sorghum bicolor (L.) Moench) brewing process

Effects of white sorghum brewing process on free amino-acids, γ-aminobutyric acid (GABA), phenolics and bioactivity, including antioxidant (by ABTS⁺ and reducing power, RP, methods), antihypertensive (angiotensin converting enzyme-I, ACE-I inhibition assay), and hypoglycaemic activity (α-glucosidase inhibition assay) were evaluated. From the wort to the beer, free amino acids decreased, but GABA and phenolics increased significantly, positively modifying the bioactive potential. ABTS and α-glucosidase inhibition activity correlated positively with at least one of the phenolic acids evaluated. Ale white sorghum beer presented high content of GABA (7.8 mg L⁻¹), phenolics (40.7 mg total phenolic acids L⁻¹), antioxidant activity (9.14 mmol Trolox equivalent L⁻¹, and 48.8 mmol ascorbic acid equivalent L⁻¹, for ABTS⁺ and RP, respectively), and exhibit ACE-I inhibition (1.0 μg captopril equivalent L⁻¹) and α-glucosidase inhibition (34.5 mg acarbose equivalent L⁻¹) activities. The level of bioactive compounds and its low ethanol content (2.3%), make beer obtained from malted white sorghum a potential functional beverage.     

Antimicrobial agent detection in ewes’ milk by the microbial inhibitor test brilliant black reduction test—BRT AiM®

The detection limits of 24 antimicrobial agents were determined in ewes’ milk by one commercially available version of brilliant black reduction test, BRT Inhibitor Test with prediffusion AiM^® (BRT AiM^®). For each drug, eight concentrations were tested on 20 milk samples from individual ewes. The detection limits of the BRT AIM^® method were determined by means of logistic regression models: 6 μg kg⁻¹ amoxycillin, 6 μg kg⁻¹ ampicillin, 51 μg kg⁻¹ cloxacillin, 2 μg kg⁻¹ penicillin “G”, 230 μg kg⁻¹ cefadroxil, 1330 μg kg⁻¹ cephalosporin “C”; 270 μg kg⁻¹ cephalexin, 92 μg kg⁻¹ cefoperazone, 120 μg kg⁻¹ ceftiofur, 69 μg kg⁻¹ cefuroxime, 6000 μg kg⁻¹ streptomycin, 1200 μg kg⁻¹ gentamycin, 3700 μg kg⁻¹ neomycin, 630 μg kg⁻¹ erythromycin, 120 μg kg⁻¹ tylosin, 390 μg kg⁻¹ doxycycline, 5500 μg kg⁻¹ oxytetracycline, 6200 μg kg⁻¹ tetracycline, 5400 μg kg⁻¹ sulfadiazine, 3200 μg kg⁻¹ sulfamethoxazole, 6500 μg kg⁻¹ sulfamethoxypyridazine, 6200 μg kg⁻¹ sulfaquinoxaline, 22000 μg kg⁻¹ chloramphenicol and 4100 μg kg⁻¹ trimethoprim. The BRT AiM^® method presents detection limits for β-lactam antibiotics that are similar to those obtained as Maximum Residue Limits (MRLs) according to Regulation 2377/90 EEC as set out by the European Union. However, for other antimicrobial agents the estimated limits were higher than those of the EU-MRLs. It is therefore advisable to enhance the sensitivity of the method for the detection of the different antimicrobial groups or to develop a combined system of different microbiological inhibitor tests that would enable the detection of a greater number of antimicrobial agents.