Bimlesh Mann

Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review

Glycomacropeptide (GMP) is a C-terminal part (f 106–169) of kappa-casein which is released in whey during cheese making by the action of chymosin. GMP being a biologically active component has gained much attention in the past decade. It also has unique chemical and functional properties. Many of the biological properties have been ascribed to the carbohydrate moieties attached to the peptide. The unique set of amino acids in GMP makes it a sought-after ingredient with nutraceutical properties. Besides its biological activity, GMP has several interesting techno-functional properties such as wide pH range solubility, emulsifying properties as well as foaming abilities which are shown to be promising for applications in food and nutrition industry. These properties of GMP have given new dimension for the profitable utilization of cheese whey to the dairy industry. A number of protocols for isolation of GMP from cheese whey have been reported. Moreover, its role in detection of sweet/rennet whey adulteration in milk and milk products has also attracted attention of various researchers, and many GMP-specific analytical methods have been proposed. This review discusses the chemico-functional properties of GMP and its role in the detection methods for checking cheese or sweet whey adulteration in milk. Recent concepts used in the isolation of GMP from cheese whey have also been discussed.摘要糖巨肽 (GMP) 是由κ-酪蛋白的C-末端部分 (f106-69)构成的一种多肽,是在凝乳酶的作用下从干酪制作过程中的乳清中释放出来。过去十年来,糖巨肽作为一种生物活性成分,引起了人们广泛的关注。它有其独特的化学和功能特性。GMP 的大多数生物特性应归因于与肽连接的糖基。GMP 中独特的氨基酸组成使它作为保健食品很受欢迎。GMP 除了具有生物活性外,它还具有使人们感兴趣的加工特性,如较广 pH 范围的溶解性、乳化性和起泡性,这些特性使GMP在食品和营养方面有较广阔的应用前景。GMP 的这些特性给人们一种全新的思维,那就是将干酪乳清合理的运用于乳制品工业中。很多关于从干酪乳清中分离GMP的实验方案和计划被报道。然而, GMP 在检测奶中甜乳清/凝乳乳清掺假方面的作用也引起了很多研究工作者的广泛关注,提出了很多分析 GMP 的具体方法。本综述讨论了 GMP 的化学和功能特性以及在干酪和甜乳清掺假中的作用,探讨了近期从干酪乳清中分离 GMP 的新的想法。

Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk

Molecular imprinted polymer (MIP) against cephalexin was synthesized by co-polymerization of functional monomer, cross-linker, radical initiator, along with target molecule (cephalexin) in a porogenic material. Binding of cephalexin towards prepared MIP was studied in different solvents (water, methanol, 1M NaCl, acetone and acetonitrile) and best binding was observed in methanol. Partition coefficient and selectivity of prepared imprint and non-imprint was also studied. Cross reactivity in terms of binding efficiency was also assessed with other antibiotics. Chromatographic study of MIP was carried out by packing prepared imprint into glass column. MIP was used as matrix in solid phase extraction (SPE) for recovery of cephalexin from spiked milk samples for further estimation by high performance liquid chromatography. No interference was observed from milk components after elution of cephalexin from MIP, indicating selectivity and affinity of MIP. On the other hand, interference was observed in eluate obtained from C18 SPE column.

Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb.

Colorimetric determination of melamine in milk using unmodified silver nanoparticles

Melamine is nitrogen rich chemical compound used as an adulterant in dairy products by unscrupulous people to increase the apparent protein content. This incident prompted the researchers to develop simple methods for easy detection of melamine in food samples. In the present paper, we report a simple and sensitive colorimetric method for detection of melamine in milk based on silver nanoparticles. This method relies upon the principle that melamine causes the aggregation of silver nanoparticles, resulting in abrupt color change from yellow to red under optimized conditions. The concentration of melamine in adulterated sample can be quantitated by monitoring the absorption spectra of silver nanoparticles using ultraviolet-visible (UV-Vis) spectrometer. The present colorimetric method which utilizes silver nanoparticles of 35 nm can reliably detect melamine down to a concentration of 0.04 mg l(-1).

Ameliorative potential of whey protein hydrolysate against paracetamol-induced oxidative stress.

The aim of the present study was to validate the antioxidant effect of whey protein hydrolysate (WPH) using a small animal model. Paracetamol is common drug that is safe at therapeutic levels; however, an overdose causes oxidative stress, which may lead to potential hepatic and renal necrosis. The protective effect of WPH against paracetamol-induced hepato-nephrotoxicity in mice was investigated in this study. The WPH was prepared by hydrolyzing ultrafiltered retentate of mozzarella cheese whey with commercial food-grade alcalase; the resulting WPH had substantial in vitro antioxidant activity. Male albino mice were treated with WPH for 4 d [intraperitoneally at 4 mg/kg of body weight (BW) per day or orally at 8 mg/kg of BW per day] before or after oral administration of paracetamol (300 mg/kg of BW) for 2 d. Two control groups were used; the negative control mice were administered water only; the paracetamol group was administered paracetamol at 300 mg/kg of BW but received no WPH. Levels of different marker enzymes (glutamate pyruvate transaminase and alkaline phosphatase), creatinine, and blood urea nitrogen were measured in the experimental animal blood sera. The WPH successfully mitigated the increase in the concentration of oxidative biomarkers such as glutathione pyruvate transaminase, alkaline phosphatase, and creatinine, and restored the level of blood urea nitrogen to normal in sera of mice in which oxidative stress was induced with an overdose of paracetamol. Furthermore, the indices of different antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, and lipid peroxidation end-products were determined in liver homogenate. The mice that were given WPH, either intraperitoneally or orally, showed increased activities of antioxidant enzymes and a reduction in thiobarbituric acid reactive substances (TBARS) compared with the paracetamol control group. The protective effect of WPH was less when administered orally than intraperitoneally. We concluded that WPH is the potential protector against paracetamol-induced hepato-nephrotoxicity and can be effectively used in health-promoting foods as a biofunctional ingredient.

Production and characterisation of whey protein hydrolysate having antioxidant activity from cheese whey

BACKGROUND Cheese whey is a rich by-product in nutritional terms, possessing components with high biological value, excellent functional properties, and an inert flavour profile. In the present study, mozzarella cheese whey was ultra-filtrated to remove lactose and mineral. The retentate was hydrolysed with food-grade enzyme alcalase and the hydrolysis conditions (pH, temperature and time) were optimised for maximum antioxidant activity using response surface methodology. RESULTS Whey protein hydrolysed for 8 h at pH 9 and 55 °C showed a maximum antioxidant activity of 1.18 ± 0.015 µmol Trolox mg(-1) protein. The antioxidant peptides were further enriched by ultra-filtration through a 3 kDa membrane. Seven peptides - β-Lg f(123-131), β-Lg f(122-131), β-Lg f(124-131), β-Lg f(123-134), β-Lg f(122-131), β-Lg f(96-100) and β-Lg f(94-100) - were identified by LC-MS/MS in the 3 kDa permeate of the hydrolysate. The incorporation of whey protein hydrolysate (WPH) in lemon whey drink (5-10 g L(-1)) increased the antioxidant activity from 76% to 90% as compared to control. CONCLUSION Hydrolysis of ultra-filtrated retentate of whey can be an energy- and cost-effective method for the direct production of WPH from whey compared to the industrial production of WPH from whey protein concentrate. This study suggests that WPH with good nutritional and biological properties can be effectively used in health-promoting foods as a biofunctional ingredient.

ACE-Inhibitory Activity of Cheddar Cheeses Made with Adjunct Cultures at Different Stages of Ripening

In this study, cheddar cheeses made with adjunct cultures L. casei ssp. casei 300 (CCA) and L. paracasei ssp paracasei 22 (CCB) were assessed for ACE inhibitory activity at different stages of ripening. ACE-inhibitory activity of the UF permeates of WSE of all the cheeses increased significantly (P<0.05), especially after the first two months of ripening. The inhibitory activity continued to increase in the cheeses made with adjunct cultures CCA (IC50 value 0.16 mg.ml-1) and CCB (IC50 value 0.20 mg.ml-1) up to third month and in CCC increased to this level after fourth month of ripening (IC50 value 0.20 mg.ml-1). The electrophoretic and RP-HPLC profiles indicated that the rate of degradation of proteins resulted in formation of smaller peptides which were higher in cheese made with adjunct cultures as compared to control cheese The result also indicated that cheeses with the addition of adjunct had higher ACE-inhibitory activity than control cheese.

Bioactive Peptides in Yogurt

Abstract Yogurt is a coagulated milk product obtained from lactic acid fermentation by the action of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. Proteolytic activity of S. thermophilus and L. delbrueckii ssp. bulgaricus indicate that both organisms possess different exopeptidases and peptidases. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health. At present, milk proteins are considered the most important source of bioactive peptides. Bioactive peptides may affect the major body systems—namely, the cardiovascular, digestive, endocrine, immune, and nervous systems. The strain should not be too proteolytic and must have the right specificity to give high concentrations of active peptides. The concentration of bioactive peptides appears to rely on a balance between their formation and further breakdown that in turn depends on storage time and conditions.

Process optimisation for preparation of caseinophosphopeptides from buffalo milk casein and their characterisation.

Caseinophosphopeptides (CPPs) are multifunctional bioactive peptides containing phosphorylated seryl residues in their sequence. In the present study, method for the production of CPPs from buffalo milk casein was optimised and characterised for their sequence, calcium solubilising and calcium binding activities. Response surface methodology was used to optimise the conditions for hydrolysis of buffalo casein by trypsin to obtain maximum yield of CPPs. The optimum hydrolysis conditions were as follows: hydrolysis pH 7.5, temperature 37 °C, hydrolysis time 7.0 h. Under these conditions, the experimental yield obtained was 10.04±0.24%, which is slightly lower than value predicted by the model. These CPPs were able to solubilise 1.03±0.08 mg la/mg CPPs in presence of excess phosphate and bind 0.935 mg of Ca/mg of CPPs. Eight phosphopeptides, i.e., α(s1)-CN f (37-58) 2P; α(s1)-CN f (37-58) 3P; α(s1)-CN f (35-58) 2P; α(s1)-CN f (35-58) 3P; α(s2)-CN f (2-21) 4P; α(s2)-CN f (138-149) 1P; β-CN f (2-28) 4P and β-CN f (33-48) 1P were identified by LC-MS/MS which contained motif for binding of divalent minerals. The sequences of these CPPs differed from that of derived from bovine casein.

Bioactive Peptides from Whey Proteins

Abstract Whey forms a rich source of biologically active components, in particular, its protein fraction is known to encompass many health-promoting effects. Whey protein derived biologically active peptides are currently the subject of intensive research. These bioactive peptides are the sequence of amino acids that are encrypted within the primary structure of milk proteins, requiring proteolysis for their release from the precursor. These are released during enzymatic digestion in-vitro or in-vivo and by microbial fermentation. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive, antioxidative and opioid-like properties etc. These functions relate to general health conditions or a reduced risk of certain chronic diseases. To ascertain the suitability of these peptides/peptides riched ingredients for functional food applications, clinical studies are required to assess their bioavailability, health claims and safety of these bioactives.