Interaction study of aflatoxin M1 with milk proteins using ATR-FTIR.

Aflatoxin M1 (AFM1) is a metabolite of carcinogenic aflatoxin B1 (AFB1) and appears in milk of dairy animals on ingestion of feed contaminated with AFB1. It has been reported to have affinity towards milk proteins, the exact mechanism of which is still unknown. In the present study, ATR-FTIR coupled with chemometrics is utilized to understand AFM1 interaction with milk proteins. The second order derivative spectra of the spectral window 1700-1600 cm-1 confirms the affinity of AFM1 towards milk proteins. The results of principal component analysis suggested that spectral window of 1700-1600 cm-1 is informative and provides an indication of the conformational changes brought by AFM1 in the secondary structure of milk proteins. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05587-x.

Methods to differentiate between cotton tract area ghee and cotton seed oil adulterated ghee.

Ghee, the clarified butter fat is one of the principal dairy products in India. In some places of India, cotton seed is fed extensively to dairy animals which changes the physico-chemical constants and fatty acid profile of the milk fat. Ghee is often adulterated with cotton seed oil and is marketed as cotton tract area ghee. Physico-chemical constants like RM value, Polenske value, BR reading, saponification value, iodine value and colorimetric tests i.e., Halphen and DPPH radical test were employed to differentiate ghee adulterated with cotton seed oil and that from cotton tract area. Chromatographic techniques like HPLC and GC-MS were also explored. Physico-chemical constants were not useful to differentiate the two types of ghee. Cyclopropenoic acids were observed only in ghee adulterated with cotton seed oil and not in cotton tract ghee. The RP-HPLC could able to distinguish the cotton tract area ghee from ghee adulterated with cotton seed oil on the basis of presence of ß-sitosterol in the latter. Halphen test was positive for cotton tract ghee, but not for the cotton seed oil adulterated ghee. Methylene blue reduction and DPPH radical test were also found to be useful to distinguish both types of ghee.

Migration study of chemical additives from low density polyethylene (LDPE) into dahi.

Dahi is widely used fermented milk product in India. Low Density Polyethylene (LDPE) is the most extensively used packaging material for Dahi in India. The present study was conducted to develop the analytical methods for extraction and migration of chemical additives from LDPE into dahi. Characterization of dahi packaging materials collected from five different firms was done by Fourier Transform Infrared Spectroscopy. Focused ultrasound solid liquid extraction method was observed to be better as compared to solid liquid extraction method as the former extracted maximum additives from the LDPE. Out of total 76 chemical additives extracted from LDPE, only eight (10.52%) matched with the existing positive list of polyolefins prescribed by Bureau of Indian Standads (BIS). The overall migration of chemical additives from all the LDPE samples was below their maximum limit as given by BIS standards. Chemical additives which migrated into the simulants included the antioxidants, fatty acids and their derivatives, unreacted hydrocarbons, plasticizers, lubricants and surfactant etc.

Physico-chemical characterization of ultrasound assisted clove oil-loaded nanoemulsion: As enhanced antimicrobial potential.

Clove oil has a high eugenol content, making it an effective antimicrobial essential oil; nevertheless, its low water solubility, high volatility, and organoleptic qualities limit its use in food systems. As a result, we created an antibacterial system using clove oil-in-water nanoemulsion. Clove oil nanoemulsions were produced using whey protein concentrate (0.1-1%) as an emulsifier by ultrasonication and various physico-chemical characteristics (stability, particle size, zeta-potential, and poly dispersity index) were investigated. Mean particle size, zeta potential and polydispersity index of the most stable nanoemulsion were 279.0 ± 8.43 nm, -34.5 ± 0.12 mV, and 0.179 ± 0.012, respectively. Most stable nanoemulsion was fairly stable at different processing parameters such as various pH (3.0 - 7.0), temperature ranges (63 - 121 °C), and ionic strengths (0.1 - 1.0 M NaCl). Finally, antimicrobial activities, such as minimum inhibitory concentration was found with 50 µL, whereas minimum bactericidal concentration was observed to be 90 µL after 8 h contact time, against E. coli and B. subtilis strains.

Effect of incorporation of iron-whey protein concentrate (Fe-WPC) conjugate on physicochemical characteristics of dahi (curd).

Dahi samples were prepared from milk incorporated with spray-dried iron-whey protein concentrate (Fe-WPC) conjugate and ferrous sulfate (FeSO4) with three different concentrations of iron i.e. 15, 20 and 25 mg/L and their quality characteristics were determined. Fe-WPC conjugate incorporated dahi showed better sensory, textural and physical attributes as compared with those of FeSO4 fortified and control dahi. Non-significant (p > 0.05) changes were observed in attributes like acidity and flavor, color and appearance, body and texture scores of dahi fortified with Fe-WPC conjugate with upto 20 mg/L iron as compared to those of control. In contrast, definite metallic flavor was perceptible in case of FeSO4 incorporated dahi even at 15 mg/L level. Water holding capacity, viscosity and firmness were significantly (p < 0.05) higher in 20 mg/L Fe-WPC conjugate incorporated dahi samples as compared with those of 20 mg/L FeSO4 incorporated dahi samples. In vitro bio accessibility of iron from Fe-WPC conjugate incorporated dahi was found to be significantly (p < 0.05) higher than that from FeSO4 incorporated dahi. Therefore, the results indicated that Fe-WPC conjugate can be fortified in dahi with upto 20 mg/L without significantly altering its physicochemical properties and with a higher bioaccessibillity of iron.

Preparation and characterization of iron-chelating peptides from whey protein: An alternative approach for chemical iron fortification.

Iron fortification of staple food is a strategy utilized worldwide to address the concern of dietary iron deficiency. However, traditional salt-based fortification methods have limitations with gastrointestinal stability and bioavailability. Iron chelating peptides from easily available and scalable proteins such as whey protein have been proposed as promising candidates to circumvent the above mentioned limitations by enhancing iron absorption and bioavailability. In this study, we report methods to produce whey protein derived iron-chelating peptides and describe their physicochemical characteristics. Peptides derived from whey proteins prepared by ultrafiltration of whey followed by hydrolysation were iron chelated to produce peptide-iron complexes. These complexes had a size of 422.9 ± 3.41 nm, chelated iron content of 36.42 µg/ mg protein, and a low zeta potential (-10.80 mV) compared to whey peptides. Spectra analysis using ultraviolet-visible absorption and Fourier transform infrared spectroscopy showed structural transformation indicating iron chelation. Mass spectrometric analysis using LC-MS/MS confirmed the presence of both hydrophilic and hydrophobic peptides in the complexes with sizes ranging from 275 Da to 1916 Da. Furthermore, reduction in the antioxidant property of peptides following iron complexing indicates iron chelation. Our results suggest that whey protein derived peptide-iron complexes can be used as a potential alternative for chemical iron fortificants for food products and also as iron supplements.

Adulteration of cow's milk with buffalo's milk detected by an on-site carbon nanoparticles-based lateral flow immunoassay.

A competitive lateral flow immunoassay using amorphous carbon nanoparticles (CNPs) and non-immunoglobulin antigen has been developed for the rapid detection of adulteration of cow's milk with buffalo's milk. Purified polyclonal antibodies against a specific buffalo's milk protein fraction were conjugated to CNPs and sprayed on a conjugate pad. The test line consisted of buffalo's skimmed milk proteins (1.6 µg/cm), while the control line contained anti-rabbit antibodies raised in goat (0.5 µg/cm). In the test procedure milk sample is mixed with 100 mM borate buffer (pH 8.8 containing 1% BSA and 0.05% Tween 20) and pipetted onto the sample-cum-conjugate pad. A black/grey test line can be observed if the sample is free from buffalo's milk. The sensitivity of the test i.e. no visible test line is 5% adulteration of cow's milk with buffalo's milk. The test has applicability at the milk receiving stations and can be applied to heated milk samples.

Distinction between glycomacropeptide and ß-lactoglobulin with 'stains all' dye on tricine SDS-PAGE gels.

Identification of glycomacropeptide (GMP) and ß-lactoglobulin (ß-lg) present in cheese whey is difficult on SDS-PAGE due to their close proximity during electrophoresis and poor sensitivity of commonly used staining dye 'coomassie brilliant blue' (CBB) towards GMP. A simple method has been developed for the detection of GMP and ß-lg by staining acrylamide gel after tricine SDS-PAGE using cationic 'stains all' dye. After staining and destaining major whey proteins, viz. ɑ-lactalbumin (ɑ-la) and ß-lg appear red while GMP stains blue. The method can be used for the identification of these macromolecules in cheese whey and the detection of adulteration of milk with rennet whey.

Impact of sequential enzymatic hydrolysis on antioxidant activity and peptide profile of casein hydrolysate.

ABSTRACT: This paper shows the potential of dual enzyme approach on antioxidant activity of casein hydrolysates. Casein was hydrolysed using the proteolytic enzymes alcalase, flavourzyme in isolation and in sequential order. Casein hydrolysates were evaluated for the degree of hydrolysis, antioxidant activity, molecular weight distribution patterns and peptide sequence. Casein hydrolysate produced by the sequential hydrolysis of alcalase and flavourzyme showed higher degree of hydrolysis and antioxidant activity as compared to hydrolysate obtained by individual enzymes. In size exclusion chromatograph of casein hydrolysate S3, peptides with molecular weight of 0.57 kDa share 12% area in total area of chromatogram which was 10 times higher than that of hydrolysate S1 and nearly half of that of hydrolysate S2. On subjecting to HPLC-TOF-ESI separation potential antioxidant peptides were identified. The peptide sequence VLPVPQ along with potential fragments was identified in hydrolysate S1 and S2 and HPHPHLS along with its potential sequence was identified in hydrolysate S1, S2 and S3. Sequential hydrolysis of casein showed better antioxidant activity and peptide profile in less duration as compared to the casein hydrolysate obtained by individual enzyme.

Assessment of physico-chemical changes in UHT milk during storage at different temperatures.

This research communication describes enzymatic and physico-chemical changes during storage of UHT milk. The UHT milk sample was stored at 5 and 30°C for 4 months and analyzed regularly at intervals of one month. During storage of UHT milk, there was a significant (P < 0.001) increase in non-protein nitrogen, non-casein nitrogen, soluble calcium, soluble magnesium and proteolysis, while a significant (P < 0.001) decrease in pH was observed. There was a slight change in the particle size and zeta potential of casein micelles. Changes were more pronounced in milk samples stored at 30°C than in those stored at 5°C. During storage, there occurred changes in pH, viscosity, salt balance and nitrogenous components which adversely affected quality. It was concluded that the proteolysis led to the acidification which had a destabilizing effect on the milk.

Encapsulation of grape seed extract phenolics using whey protein concentrate, maltodextrin and gum arabica blends.

Grape seed extract (GSE) contain phenolic compounds that decrease the proclivity to various chronic diseases such as several types of cancer and cardiovascular diseases. The objective of the present study was to investigate the encapsulation of GSE polyphenols and their characterization. For this study, whey protein concentrate (WPC), maltodextrin (MD) and gum arabic (GA) were evaluated as encapsulating materials. For the preparation of stable microcapsules different WPC:MD/GA (5:0, 4:1, 3:2 and 0:5) ratios were assessed using ultrasonication for different time periods (20-40 min) followed by freeze drying. Encapsulation efficiency, antioxidant activity, particle size, surface morphology and release mechanism were determined. The GSE microcapsules coated with WPC:MD/GA ratio of 4:1 and 3:2 with core to coat ratio of 1:5 and prepared by sonication for 30 min were found to have highest encapsulation efficiency (87.90-91.13%) and the smallest particle size with maximum retention of antioxidant activity. Under optimized conditions, the low level release (43-49%) of phenolic compounds resulted under simulated gastric condition and significantly (p < 0.05) increased (88-92%) under simulated intestinal condition. Thus the results indicated blending of MD or GA with WPC improved the microencapsulation of GSE.

Development and validation of an analytical method for determination of bronopol and kathon preservative in milk.

The bronopol and kathon are chemical preservative which prevent degradation of milk samples and maintain authenticity in analysis. The detection is based on HPLC-UV-Vis spectroscopy, in which C18 column (250 mm × 4.6 mm, 5 µm) was used for chromatographic separations, with a mobile phase comprising 0.1% phosphoric acid in water: Methanol: 0.1% phosphoric acid in acetonitrile (80:10:10) at a flow rate 0.8 ml/min at ambient temperature and with the UV detection at 250 nm for bronopol and 274 nm for kathon. The retention time of bronopol, kathon (MI 2-methyl-4-isothiazolin-3-one) and kathon (CMI 5-chloro-2-methyl-4-isothiazolin-3-one) was 4.52 min, 3.98 min and 6.68 min respectively with a total run time of 10 min. The linearity of the method was satisfactory with regression coefficient (R2) = 0.99. The limit of quantification was 72, 240, 390 mg L-1 for bronopol, kathon (MI) and kathon (CMI) respectively. Five spiked levels (62.5, 125, 250, 500 and 1000 mg L-1) were used to determine the recovery of bronopol, kathon (MI) and kathon (CMI) which was found to be 95.41 ± 11.84, 95.75 ± 8.21 and 92.22 ± 14.64% respectively, with relative standard deviations in the range 5.9-14.6%. The standardized analytical method was successfully used to rapidly detect bronopol and kathon in milk samples.

Estimation of steviol glycosides in food matrices by high performance liquid chromatography.

Steviol glycosides (SGs) are non-caloric, natural sweetener obtained from plant Stevia rebaudiana and are used as sugar substitute in foods. The level of SGs in foods should not exceed maximum permissible limit defined by regulatory agencies. Thus analytical methods are required for assay of stevioside (Stev) and rebaudioside A (Reb A), which are two major constituents of SGs, in foods. A method for extraction of Stev and Reb A from dairy viz., flavoured milk, flavoured yoghurt and non-dairy foods viz., carbonated water, jam, chewing gum and estimation of these by HPLC has been described. Extraction of SGs from dairy samples was achieved by treating samples with 20% acetonitrile in presence of Carrez solutions while these can be simply extracted with water from non-dairy samples. Separation and estimation of these two glycosides was achieved on C18 column (length: 4.6 × 250 mm, particle size: 5 µm) using isocratic mobile phase prepared by mixing of acetonitrile and 10 mM sodium phosphate buffer (pH 2.6) in ratio of 32:68 (v/v). Recovery of two SGs was quantitative. Separation and estimation of SGs by HPLC was robust. Limit of detection and limit of quantitation for Reb A in different food was in range from 1.057-1.834 to 3.525-6.114 mg kg-1 while that of Stev was from 1.679-2.912 to 5.596-9.707 mg kg-1, respectively. Neotame, an artificial sweetener can be used as internal standard for separation of SGs.

Physico-chemical and antimicrobial properties of d-limonene oil nanoemulsion stabilized by whey protein-maltodextrin conjugates.

In the present investigation d-limonene oil (4-isopropenyl-1-methylcyclohexene) was encapsulated by ultra-sonication method using whey protein (WP)-maltodextrin (MD) conjugates as coating material and their characterization was done with respect to physico-chemical and antimicrobial properties. Antimicrobial activity of limonene oil (LO) nanoemulsion and bulk LO dissolved in dimethyl sulphoxide (DMSO) were assessed by agar well diffusion method. Stable formulation of d-limonene oil nanoemulsion [5.0% LO + 9.0% WP-MD (1:2 w/w) conjugate] had shown mean particle size, zeta potential and poly dispersity index of 116.60 ± 5.30 nm, - 19.64 ± 0.23 mV and 0.205 ± 0.02 respectively. LO nanoemulsion were stable to different food processing conditions like heat treatments, ionic strength (0.1-1.0 M) and pH (3.0-7.0). LO nanoemulsion was stable for 15 days at 25 °C and it had shown particle size of 332.20 ± 5.40 nm at 15th day. It was observed that minimum inhibitory concentration (MIC) of both LO nanoemulsion and bulk LO dissolved in DMSO were at 12.50 µl/ml against Bacillus cereus (ATCC 14459), Escherichia coli (ATCC 25922), Enterococcus faecalis (NCDC 115) and Salmonella typhi (NCDC 6017). Since d-limonene has been considered to be a safer alternative compared to synthetic antimicrobial food additives, the present investigation will be helpful in developing a more effective antimicrobial system for the production and preservation of foods.

Construction of a lateral flow strip for detection of soymilk in milk.

A lateral flow based detection method for ascertaining the presence of soymilk in whole bovine milk has been described. The method uses commercially available rabbit anti-soy protein antibodies conjugated to gold nanoparticles (AuNPs) wherein soymilk protein in adulterated milk and soymilk protein at test line competes for limited antibodies. At control line, anti-rabbit immunoglobulin was immobilized for ensuring flow properties of antibody-conjugated AuNPs. Absence or diminished intensity of band at test line indicates presence of soymilk in milk. The soymilk detection limit was 1.75% (v/v) in whole bovine milk and results are available in 5 min. Constructed lateral flow device can be used for on-spot examination of soymilk in milk.

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.

Encapsulation of antioxidant peptide enriched casein hydrolysate using maltodextrin-gum arabic blend.

Antioxidant peptide enriched casein hydrolysate (AO-CH) are receiving increasing attention due to their potential as functional ingredient. Encapsulation of AO-CH using maltodextrin-gum arabic (MD/GA) as wall material could represent an attractive approach to overcome the problems related to their direct application. Encapsulation parameter were optimized using different ratio of core to coat and proportion of coating material (10:0, 8:2, 6:4) under varying pH (2-8) for encapsulation efficiency (EE).The preparation P3 resulted in maximum EE (87%) using core to coat ratio 1:20, at pH 6.0 with 8:2 MD/GA ratio. The encapsulated preparation showed reduced bitterness (p < 0.05) compared to the casein hydrolysate together with maximum retention of antioxidant activity (93%). Further, the narrow range of particle size, indicates their better stability and represents a promising food additive for incorporation in food.

Formulation and characterization of nanoencapsulated curcumin using sodium caseinate and its incorporation in ice cream.

In the present investigation, the preparation and characterization of a curcumin nanoemulsion with milk protein (sodium caseinate) and its incorporation into ice cream were undertaken. Among the different combinations, the most stable formulation was observed using milk fat (8%), medium chain triglycerides (2%), curcumin (0.24%) and sodium caseinate (6%) with a mean particle size of 333.8 ± 7.18 nm, a zeta potential of -44.1 ± 0.72 mV and an encapsulation efficiency of 96.9 ± 0.28%. The effect of different processing conditions (heating, pH and ionic strength) on the particle size distribution and zeta potential of the nanoemulsion was evaluated. During heat treatment, the particle size of the nanoemulsion was increased from 333.8 ± 7.18 to 351.1 ± 4.04 nm. The nanoemulsion was destabilized at pH 4.6 and the particle size increased above and below pH 5.0. However, there was a slight increase in the particle size with a change in the ionic concentration. The release kinetics data suggested that in simulated gastro-intestinal digestion, the nanoemulsion was stable against pepsin digestion (a 5.25% release of curcumin), while pancreatic action led to a 16.12% release of curcumin from the nanoemulsion. Finally, our formulation was successfully incorporated into ice cream and the sensory attributes were evaluated. No significant difference was observed in the scores of the sensory attributes between the control and ice cream prepared with a curcumin nanoemulsion. Moreover, the encapsulation efficiency of the curcumin incorporated into the ice cream was 93.7%, which indicates that it can withstand the processing conditions. The findings suggest that ice cream is a suitable dairy product for the delivery of lipophilic bioactive components (curcumin) which can be used for therapeutic purposes.

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).

Antioxidant activity of whey protein hydrolysates in milk beverage system.

The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of all the hydrolysates i.e. flavourzyme (0.81 ± 0.04), alcalase (1.16 ± 0.05) and corolase (1.42 ± 0.12) was higher than the WPC (0.19 ± 0.01). Among these, whey protein hydrolysates prepared using corolase showed maximum antioxidant activity. Total 15 ß-lactoglobulin, 1 α-lactoalbumin, and 6 ß-casein derived peptide fragments were identified in the WPHs by LC-MS/MS. Due to their size and characteristic amino acid composition, all the identified peptides may contribute for the antioxidant activity. The strawberry and chocolate flavoured milk was supplemented with WPC and WPHs and 2 % addition has shown increase in antioxidant activity upto 42 %. The result suggests that WPH could be used as natural biofunctional ingredients in enhancing antioxidant properties of food products.