Oleoylation of sugarcane bagasse hemicelluloses using N‐bromosuccinimide as a catalyst

Conditions for the preparation of esters of sugarcane bagasse hemicelluloses with oleoyl chloride using N‐bromosuccinimide (NBS) as a catalyst in the N,N‐dimethylformamide/lithium chloride system under mild conditions were studied comparatively. The oleoylation was followed in terms of yield and degree of substitution (DS). The parameters optimised included oleoyl chloride concentration as the molar ratio of oleoyl chloride/anhydroxylose units in native hemicelluloses (1:1–8:1), NBS concentration (0.5–3.0%), reaction time (20–100 min) and reaction temperature (30–100 °C). Results were also compared with those for other catalysts such as H₂SO₄ and four tertiary amine catalysts, pyridine, 4‐dimethylaminopyridine, N‐methyl pyrrolidine and N‐methyl pyrrolidinone. An oleoyl chloride/hemicellulose molar ratio of 3:1 was found to be necessary for the reaction to proceed smoothly and to yield a product with a low DS. The new materials were characterised by FT‐IR and ¹H and ¹³C NMR spectroscopies as well as thermal analysis. The thermal stability of the oleoylated hemicelluloses decreased slightly upon chemical modification, but no significant further decrease in thermal stability was observed for DS ≥ 0.29. Copyright © 2004 Society of Chemical Industry     

Synthesis of high fatty acid starch esters with 1‐butyl‐3‐methylimidazolium chloride as a reaction medium

In this work, high fatty acid esters of corn starch were synthesized by reacting the starch with fatty acid methyl ester using 1‐butyl‐3‐methylimidazolium chloride (BMIMCl) ionic liquid (IL) as reaction media. The effect of reaction variables such as the catalyst amount, molar ratio of fatty acid methyl ester/anhydroglucose unit (AGU) in starch, pyridine/AGU molar ratio, reaction temperature, as well as reaction time on the degree of substitution (DS) of starch esters was investigated. The experimental results showed that the DS value of the obtained starch esters could be varied depending on the process conditions. In the optimum reaction condition, the achieved maximum DS of starch laurate and starch stearate was 0.37 and 0.28, respectively, at a reaction temperature of 110°C for starch laurate and 120°C for starch stearate for a reaction duration of 2 h. Furthermore, the starch esters were characterized by FTIR, SEM, and X‐ray diffractometry (XRD) techniques, respectively. Results from FT‐IR spectroscopy suggested that the hydroxyl groups in the starch molecules were converted into ester groups. SEM and XRD studies showed that the morphology and crystallinity of starch esters were disrupted largely in the IL medium under the reaction conditions.     

An improved method for the measurement of 3‐monochloropropanediol esters by matrix solid‐phase dispersion supported liquid–liquid extraction

3‐Monochloropropanediol (3‐MCPD) esters are contaminants produced from the high‐temperature processing of edible oils. The accurate measurement of 3‐MCPD using an easy‐to‐follow and reliable method that uses a readily available instrument is important. Here, we report an acid transesterification heptafluorobutyrylimidazole (HFBI) derivatisation method for the accurate measurement of 3‐MCPD esters in edible oils. We developed a dispersed matrix solid‐phase supported liquid–liquid extraction (DMSP‐SLE) system to remove impurities. Both the transesterification and DMSP‐SLE conditions were optimised. A good linear relationship was obtained within the range of 0.05–10 mg kg^?1 (R² ≥ 0.999) in both blank solvent and an oil sample. The limit of detection was 20.36 μg kg^?1. The average recovery of the 3‐MCPD esters spiked at 0.5, 1.0 and 2.0 mg kg^?1 into a blank oil matrix was in a range from 105.09 ± 2.77% to 120.16 ± 10.88%. The method we developed was further confirmed by performing detection on a Food Analysis Performance Assessment Scheme (FAPAS) sample.     

Detection of clenbuterol in beef meat,liver and kidney by mid‐infrared spectroscopy (FT‐Mid IR) and multivariate analysis

Mid‐infrared spectroscopy (FT‐Mid IR) coupled with multivariate analysis was used to predict clenbuterol in beef meat, liver and kidney. A SIMCA model was also developed to discriminate between pure (beef meat, liver and kidney) and spiked with clenbuterol samples (beef meat‐clenbuterol, liver‐clenbuterol and kidney‐clenbuterol). The best models to predict clenbuterol concentrations were obtained using the partial least squares algorithm (PLS) with a R² > 0.9 and SEC and standard error of prediction <0.296 and 0.324, respectively. The SIMCA model used to discriminate pure and spiked with clenbuterol samples showed 100% correct classification rate. Methods detection limit was 2 μg kg^?1. FT‐Mid IR coupled with chemometrics could be a simple and rapid screening tool for monitoring clenbuterol in beef meat, liver and kidney implicated in food poisoning. This method could be use for screening purposes.     

Rapid microwave-assisted transesterification for the preparation of fatty acid methyl esters from the oil of yellow horn (<Emphasis Type="Italic">Xanthoceras sorbifolia</Emphasis> Bunge.)

An efficient microwave-assisted transesterification (MAT) technique was developed to prepare fatty acid methyl esters (FAMEs) from yellow horn (Xanthoceras sorbifolia Bunge.) oil. To evaluate the applicability of this novel MAT method, the conversion yields of FAMEs and compositions were compared with those from the standard method of transesterification (AOAC Method 996.06). The results showed that MAT had obvious advantages to standard techniques in terms of short duration, high efficiency and stable composition. The parameters influencing the MAT procedure including microwave irradiation power, temperature, reaction time, molar ratio of methanol to oil and catalyst concentration were optimized. The maximum yield of FAMEs reached 96% under optimal MAT conditions of transesterification irradiation power 500 W, temperature 60 °C, 6 min, 1% wt catalyst and a molar ratio of methanol/oil 6:1 (v/v). Validation of the MAT method was performed in terms of repeatability and reproducibility; relative standard deviations for conversion yields were lower than 4.37 and 6.83%, respectively. These results revealed that the MAT method developed in the present investigation is suitable for the sample preparation of FAMEs from plant oils.     

Characterisation of pear distillates from wild and cultivated varieties in Sardinia

The aroma fraction of Italian distillates of wild (Pyrus amygidaliformis, Vill., namely ‘Pirastru’) and cultivated (Pyrus communis, L. cvs. ‘Coscia’, ‘Precoce di Fiorano’ and ‘Butirru de Austu’) pear varieties grown in the northern part of the island of Sardinia was investigated. Distillates from fermented mashes were obtained from the same producer and, in most cases, for 3 years following the same production process. Aroma analyses were performed by GC‐FID and GC‐MS techniques with direct GC injection of full proof distillates. Differences were found in the aromatic profile of all the distillates depending on their varietal origin. Wild Pirastru pear distillates were the richest in isoeugenol (0.186 g hL^?1 a.a.), ethyl benzoate (0.388 g hL^?1 a.a.) and ethyl phenylacetate (0.12 g hL^?1 a.a.) but the poorest in 1‐hexanol (2.69 g hL^?1 a.a.), n‐hexyl acetate (0.07 g hL^?1 a.a.), Σ‐farnesene isomers (0.80 g hL^?1 a.a.) and citronellol (0.007 g hL^?1 a.a.). Pirastru distillates were also the poorest in methyl and ethyl unsaturated decanoate isomers, the so‐called Williams pear esters (0.002 g hL^?1 a.a.). On the contrary, these compounds that characterise the aroma of Williams pear distillates were abundant in Coscia spirits (1.032 g hL^?1 a.a.). The latter, together with Precoce di Fiorano, were rich in farnesene isomers (6.48 and 9.26 g hL^?1 a.a. respectively). Evaluation of the data by univariate (anova and Kruskal–Wallis) and multivariate (PCA and HCA) statistical analyses revealed that some components (the classes of acetates, farnesene isomers, methyl and ethyl unsaturated decanoate esters and citronellol) are relevant in discriminating the distillates according to variety.     

Influence of exopolysaccharide‐producing cultures on the volatile profile and sensory quality of low‐fat Tulum cheese during ripening

The objective of this investigation was to compare the composition and changes in the concentration of volatiles in low‐fat and full‐fat Tulum cheeses during ripening. Tulum cheese was manufactured from low‐ or full‐fat milk using exopolysaccharide (EPS)‐producing or non‐EPS‐producing starter cultures. A total of 82 volatile compounds were identified belonging to the following chemical groups: acids (seven), esters (21), ketones (14), aldehydes (six), alcohols (14) and miscellaneous compounds (20). The relative amounts of acids, alcohols and aldehydes increased in the cheeses made with EPS‐producing cultures during 90 days of ripening. Differences were found in the volatile profile of full‐fat Tulum cheese compared with the low‐fat variant, especially after 90 days of ripening. Exopolysaccharide‐producing cultures changed the volatile profile, and the EPS‐producing cultures including Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus + Lactobacillus helveticus (LF‐EPS2) produced cheese with higher levels of methyl ketones and aldehydes than the non‐EPS cultures. In the sensory analysis, full‐fat Tulum cheeses and the cheese produced with the EPS‐producing culture containing Lb. helveticus (LF‐EPS2) were preferred by the expert panel. It was concluded that the use of EPS‐producing starter cultures in the manufacture of low‐fat Tulum cheese had the potential to improve the flavour.     

Application of factorial design in optimization of anion exchange resin based methylation of vegetable oil and fats

A simple, rapid and fairly selective method for the preparation of fatty acid methyl esters (FAMEs) based on anion exchange resin Amberlite IRA-904 catalyzed transesterification of vegetable oil/fat with iodomethane has been described. The vegetable oil and animal fats used were sunflower oil, palm oil, vanaspati (hydrogenated vegetable oil), olive oil, tallow and butter. A Plackett–Burman factorial experimental design was used as a multivariate strategy for the evaluation of the effects of varying several variables at once. The effects of five different variables amount of resin, strength of sodium hydroxide, volume of iodomethane, heating time and temperature of thermostatic water bath, on the yield of fatty acid methyl esters (FAMEs) have been investigated. From these studies, certain variable showed up as significant, and they were optimized by a using 2³ + star central composite design, which involved 16 experiments. The best conditions for transesterification reaction were as follows: amount of resin 2 g, strength of sodium hydroxide 0.25 N, volume of iodomethane 400 µl, heating time 2 min at 70 °C. A standard IUPAC method was used to prepare FAMEs from vegetable oil/fats for comparative purpose. Finally samples of oil/fat obtained from both methods were analysed by Gas liquid chromatography. Analytical results for the FAMEs by resin based proposed method, and conventional IUPAC method showed a good agreement, thus indicating the possibility of using Amberlite IRA-904 based transesterification instead of intensive treatments inherent with the conventional time-consuming methods.

Industrial relevance

Fatty acids are the main components of edible oil and fats, therefore determination of fatty acid composition is so far one of the important parameters for quality evaluation and nutritional value determination of edible oil and fats. The analysis of fatty acid is usually carried out by Gas liquid chromatography (GLC) after conversion of volatile fatty acid methyl esters (FAME) although other ester may be prepared for specific purpose.The endeavor of present work was to improve the FAMEs preparation method, proposing the development of anion exchange resin Amberlite IRA-904 based transesterification of edible oil/fat with iodomethane as alkylating reagent. The present method besides being rapid and reproducible avoids the use of classical saponification, washing of esters and solvent extraction step.     

Effect of high‐pressure homogenisation on the retention of selected aroma compounds in model dairy emulsions

The aim of this work was to investigate the effects of high‐pressure homogenisation on the aroma retention of mixed sodium caseinate–whey protein (2% + 2% w/v) emulsions. For this purpose, raw and pasteurised emulsions, with different fat contents (5%, 8% and 15% w/v) and subjected to different homogenisation pressures (0, 18, 100 and 150 MPa), were produced. The retention of seven aroma compounds (hexanal, benzaldehyde, diacetyl, ethyl butyrate, isoamyl acetate, ethyl hexanoate and ethyl octanoate) was evaluated by static headspace gas chromatography. Results showed that aroma release was affected by the presence and the quantity of the lipid phase. As regards homogenisation, an increase in the retention of hexanal with homogenisation pressure was observed and interactions between hexanal and caseinate were suggested. Benzaldehyde showed significant changes in headspace partition with increasing pressure only in the 5% fat content emulsions, whereas no homogenisation effect on aroma retention was observed for diacetyl or esters.     

Wine authentication with Fourier Transform Infrared Spectroscopy: a feasibility study on variety,type of barrel wood and ageing time classification

Wine aging in wooden barrels improves the final products’ sensory profile and increases the price. Among the different types of woods, the International Enological Codex of the International Organisation of Vine and Wine (OIV) approves only the use of Oak and Chestnut, thus producing a need for fast and cost‐effective authentication methods. In this study Fourier Transform (FT)‐mid‐infrared (IR) spectroscopy combined with chemometrics was employed to analyze and discriminate wines aged in barrels made from different type of wood species and in stainless steel tanks, over a period of 12 months. The wines used were made from four indigenous Greek grape varieties, two white and two red. A complete differentiation of the samples was achieved according to grape variety, the container type and the aging time based on two spectral regions (from 1800 to 1500 cm^?1 and from 1300 to 900 cm^?1) of their FT‐IR spectra.     

Structural characterisation of β‐cyclodextrin crosslinked by adipic acid

This study was conducted to investigate the structural characterisation of β‐cyclodextrin (β‐CD) crosslinked by adipic acid. β‐CD was treated with different concentrations (0%, 5%, 10% and 15%, w/v) of adipic acid. Different instruments, such as scanning electron microsope (SEM), Fourier‐transform infrared (FT‐IR) spectroscopy and ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were used to find out chemical structure in the crosslinked β‐CD. SEM analysis suggested that crosslinking β‐CD with 15% adipic acid changed the original morphology and considerably increased the particle size of the raw material. FT‐IR spectroscopy data showed that an intensive absorption band at 1706 cm^?1 was present in the β‐CD samples treated with 10% and 15% adipic acid, indicating a crosslinking between hydroxyl groups of β‐CD and carboxyl groups of adipic acid. NMR spectra revealed that the ester linkages between hydroxyl groups of β‐CD and carboxyl groups of adipic acid were formed after crosslinking of β‐CD with adipic acid.     

Combined effects of a natural Allium spp. extract and modified atmospheres packaging on shelf life extension of olive‐based paste

This study aimed to extend the shelf life of non‐thermally stabilised olive‐based paste by adding different concentrations (0.2, 0.5, 1.0 g kg^?1) of a natural Allium spp. extract and/or by using different packaging atmospheres (MAP1, 75% Ar‐23% CO₂‐2% H₂; MAP2, 70% N₂‐30% CO₂) during refrigerated storage. Higher amounts of the natural extract gave the highest level of microbial inhibition, increased hardness, a greater release of aldehydes, esters, and ketones, and a better colour preservation when used with MAP1. The natural extract and MAP, either alone or in combination, produced no sensory defects (except for the control in MAP2), but a greater loss of typical olive flavour. The addition of the natural extract used with an appropriate MAP, can retain the quality attributes and extend shelf life for approximately 14 days longer than the control (without natural extract and MAP), and a better performance is achieved by using greater amounts of natural extract and MAP1.     

Storage and ultraviolet‐induced tissue stress effects on fresh‐cut pineapple

The effect of UV‐induced stress on the volatile aroma compounds in cut pineapple was compared with that of storage at 4 °C for 24 h. Eighteen volatile compounds were identified by solid‐phase microextraction (SPME) in fresh‐cut pineapple. Methyl‐2‐methylbutanoate, methyl hexanoate, methyl 5‐hexenoate, ethyl hexanoate and ethyl 5‐hexenoate were the major aroma compounds. Storage at 4 °C for 24 h, and exposure of cut fruit to UV radiation for 15 min caused a considerable decrease in the concentration of esters and increase in the relative amount of copaene. This sesquiterpene, when added to crushed cantaloupe melon (0.1 mg g^?1), inhibited microbial growth in the fruit over a period of 24 h at 20 °C. Cis‐ and trans‐ocimene were present in the fruit but their production was not photo‐induced by UV irradiation. Ocimene, however, was a potent antimicrobial agent that killed microorganisms when added to the crushed fruit and stored at 20 °C for 24 h. The results indicate that sesquiterpene phytoalexins could contribute to the defense mechanism in wounded pineapple tissue. Published in 2004 for SCI by John Wiley & Sons, Ltd.     

Characterization of volatile compounds in Fen‐Daqu – a traditional Chinese liquor fermentation starter

Fen‐Daqu is a saccharifying agent and fermentation starter for the production of Chinese liquor Fen (alcoholic spirit) and Fen traditional vinegar. The volatile compounds produced at seven incubation steps were analysed by HS‐SPME‐GC‐MS. A total of 83 major volatile compounds were identified, including 23 esters, 8 acids, 24 alcohols, 18 ketones and aldehydes, 6 pyrazines and 4 acetals. Data obtained by HS‐SPME‐GC‐MS were subjected to principal component analysis. The trajectory plots of volatile compounds in Fen‐Daqu samples obtained during successive steps of incubation were revealed. The major compounds that contributed to discrimination were hexanal, (E)‐2‐octenal, (Z)‐2‐octen‐1‐ol, nonanoic acid, 1‐octanol, 2‐decen‐1‐ol, hexyl acetate, (E)‐2‐octen‐1‐ol, acetic acid, ethyl acetate, phenylethyl alcohol, ethyl alcohol, octanoic acid, 1‐octanol, 3‐methyl‐2‐buten‐1‐ol and pyrazines. Copyright © 2012 The Institute of Brewing & Distilling     

Proximate and fatty acid compositions and sensory acceptability of Hispanic consumers towards rib‐eye steaks from forage‐finished steers

Proximate and fatty acid compositions and sensory acceptability of rib‐eye steaks (fresh and 6 months frozen‐stored) from three forage‐finished steers [S1 (bermudagrass + ryegrass, etc.); S2 (bermudagrass + ryegrass + berseem, etc.); S3 (bermudagrass+berseem+soybean+brown midrib sorghum, etc.)] and one commercial steak (C), cooked by one‐sided and/or two‐sided grilling, were evaluated. All forage‐finished steaks had lower [omega‐6/omega‐3] ratios than C. Sensory liking was assessed by Hispanic consumers. Raw C steak had higher fat and lower protein contents than others (S1, S2 and S3). Concerning raw steaks, S3 had higher liking scores for overall appearance and fat appearance than others. Two cooking methods did not cause significant differences in sensory liking. Juiciness and overall liking of cooked C and S3 (one‐sided grilling) steaks were not significantly different. Purchase intent (after knowing health benefits of forage‐finished steaks) increased from 62.0–73.8% to 69.8–85.7%. Forage‐finished steaks showed a potentially healthier lipid profile than grain‐finished steaks and had market potential towards Hispanic population.     

Rapid Differentiation of Listeria monocytogenes Epidemic Clones III and IV and Their Intact Compared with Heat‐Killed Populations Using Fourier Transform Infrared Spectroscopy and Chemometrics

The objectives of this study were to determine if Fourier transform infrared (FT‐IR) spectroscopy and multivariate statistical analysis (chemometrics) could be used to rapidly differentiate epidemic clones (ECs) of Listeria monocytogenes, as well as their intact compared with heat‐killed populations. FT‐IR spectra were collected from dried thin smears on infrared slides prepared from aliquots of 10 μL of each L. monocytogenes ECs (ECIII: J1‐101 and R2‐499; ECIV: J1‐129 and J1‐220), and also from intact and heat‐killed cell populations of each EC strain using 250 scans at a resolution of 4 cm^?1 in the mid‐infrared region in a reflectance mode. Chemometric analysis of spectra involved the application of the multivariate discriminant method for canonical variate analysis (CVA) and linear discriminant analysis (LDA). CVA of the spectra in the wavelength region 4000 to 600 cm^?1 separated the EC strains while LDA resulted in a 100% accurate classification of all spectra in the data set. Further, CVA separated intact and heat‐killed cells of each EC strain and there was 100% accuracy in the classification of all spectra when LDA was applied. FT‐IR spectral wavenumbers 1650 to 1390 cm^?1 were used to separate heat‐killed and intact populations of L. monocytogenes. The FT‐IR spectroscopy method allowed discrimination between strains that belong to the same EC. FT‐IR is a highly discriminatory and reproducible method that can be used for the rapid subtyping of L. monocytogenes, as well as for the detection of live compared with dead populations of the organism.     

Antioxidant,enzyme inhibitory and antiproliferative activity of polyphenolic‐rich fraction of commercial dry ginger powder

Polyphenolic‐rich fraction obtained from locally produced dry ginger powder in Brahmaputra valley, India, and commercially available dry ginger (Zingiber officinale) rhizome powder consisted of [6]‐gingerol (41.9%), [6]‐shogaol (24.3%), 1‐dehydro‐6‐gingerdione (8.6%), [8]‐gingerol (7.2%), [10]‐gingerol (5.1%), [6]‐paradol (5.9%) and [4]‐gingerol (3.6%). Traces of methyl‐[6]‐gingerol and methyl‐[8]‐gingerol (both at 1.8%) were also detected. The fraction exhibited high antioxidant capacity [total phenolics (TP), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA assay)], effectively inhibited isolated digestive enzymes (α‐glucosidase, pancreatic lipase and angiotensin converting enzyme) and inhibited the proliferation of colon (HT29; IC₅₀ of 1.06 ± 0.02 mg mL^?1) and gastric (AGS IC₅₀ of 1.29 ± 0.03 mg mL^?1) adenocarcinoma cells, without affecting the proliferation of their nontransformed counterparts (IC₅₀ > 2.0 mg mL^?1). This case study demonstrates that locally produced and commercially available dry ginger powder from Brahmaputra valley, India, retains numerous food components that may enhance human health.     

Electrospray MS‐based characterization of β‐carbolines – mutagenic constituents of thermally processed meat

The β‐carbolines 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson's disease and cancer. As they can be formed during the heating of protein‐rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff. For this purpose, LC‐MS and LC‐MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H‐(H^?)]⁺ and [M+H‐2H]⁺. In this study, we have investigated the formation of these ions by accurate‐mass electrospray MS/MS and demonstrated that these ions are formed from gas‐phase ion‐molecule reactions between water vapor present in the collision cell and the protonated molecule of 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC‐MS and LC‐MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of β‐carbolines is performed, as this residue may affect the reliability in the results of quantification.     

Rheological,gelling and emulsifying properties of a glycosylated and cross‐linked caseinate generated by transglutaminase

The impacts of oligochitosan glycosylation and cross‐linking on some properties of a commercial caseinate were investigated in this study. The glycosylated and cross‐linked caseinate with glucosamine content of 4.74 g kg^?1 protein was generated by transglutaminase (TGase) and oligochitosan at pH 7.5 and 37 °C, with fixed substrate molar ratio of 1:3 (acyl donor to glucosamine acceptor), caseinate content of 50 g L^?1, TGase of 10 kU kg^?1 protein and reaction time of 3 h, respectively. In comparison with the caseinate, the glycosylated and cross‐linked caseinate had decreased reactable amino groups (0.58 vs. 0.51 mol kg^?1 protein), higher apparent viscosity, decreased emulsifying activity index (about 14.5%) and statistically unchanged emulsion stability index (92.6 vs. 90.5%). Based on the mechanical spectra of the acid‐induced gels, the glycosylated and cross‐linked caseinate showed shorter gelation time (95 vs. 200 or 220 min) than the caseinate or cross‐linked caseinate. The gels prepared from the glycosylated and cross‐linked caseinate also had enhanced hardness, springiness and cohesiveness. The results indicated that TGase‐mediated oligochitosan glycosylation and cross‐linking has the potential to obtain new protein ingredients.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.