Influence of buffer and l-phenylalanine concentration in the Rhizomucor miehei lipase catalysed synthesis of l-phenylalanyl-d-glucose ester investigated through response surface methodology

Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of l-phenylalanyl-d-glucose using unprotected l-phenylalanine and d-glucose were optimized using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving 32 experiments of five variables (l-phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h and buffer concentration in mM) at five levels. A second-order polynomial equation was developed in terms of linear, quadratic and cross product terms to study the effects of variables on esterification yields. A R ² value of 0.7 was obtained for this complex reaction. From the surface and contour plots it was found that higher yields were observed for a very narrow pH range of 4.5–6.5 at l-phenylalanine concentrations above 3 mmol. The extent of esterification decreased with increase in RML concentration for incubation periods below 60 h. However, longer incubation periods above 60 h, enhanced esterification at all RML concentrations. Lower conversions below 0.5 mmol required less than 3.5 mmol l-phenylalanine concentration and a broad range of buffer concentration from 0.1 mM (0.1 ml, 0.1 M buffer of pH 6.0) to 0.5 mM (0.5 ml, 0.1 M buffer of pH 6.0). However, higher conversions from 0.6 to 0.8 mmol required a buffer concentration above 0.25 mM (0.25 ml, 0.1 M buffer of pH 6.0) at l-phenylalanine concentrations above 3.5 mmol. An optimum predicted yield of 1.01 mmol for l-phenylalanyl-d-glucose at 3 mmol l-phenylalanine, 100 mg of RML, 24 h incubation period, 0.5 mM (0.5 ml of 0.1 M buffer), pH 4.8 acetate buffer was found to agree with 0.97 mmol obtained under these experimental conditions. Validation experiments carried out under random conditions also exhibited good correspondence between predicted and experimental yields.     

Bioluminescence and ice-nucleation microbial biosensors for l-arabinose content analysis in arabinoxylans

Microbial biosensors are analytical devices converting a biochemical signal into a quantifiable response. Due to their molecular properties, they can be diversely designed in order to fulfill the needs of different fields, from food industry to environmental sciences. One of the possible applications of microbial biosensors concerns arabinoxylans, components of dietary fibers having potential as functional food ingredients, which are also used for biogas production. The aim of this study was to develop reporter strains capable to evaluate the content of l-arabinose monosaccharides in arabinoxylans. The bioluminescent strain DPDaraBAD contains a plasmid in which luxCDABE genes of Photorhabdus luminescens are inserted under the control of Escherichia coli arabinose operon promoter. The ice-nucleation-active strain NIaraBAD bears a similar fusion using the same promoter upstream of the inaZ gene of Pseudomonas syringae. Sufficient response of biosensor cells was obtained after NIaraBAD incubation at 29 °C for 3 h and DPDaraBAD for 6 h, followed by 30-min incubation with l-arabinose. Both reporter strains responded specifically and quantitatively in the presence of the inducer with a detection limit of 0.009 and 0.015 g l^?1, respectively. Validation of reporter strains was performed in comparison with the l-arabinose concentration values obtained by high-performance anion-exchange chromatography (HPAEC) and demonstrated mean divergence of 9.59 % for bioluminescent strain and 15.86 % for ice-nucleation strain, which for microbial sensors is an acceptable dissimilarity. These l-arabinose microbial biosensors can be used as an alternative tool to chromatographic methods in monosaccharide content analysis of arabinoxylans.     

Development of an l-rhamnose bioluminescent microbial biosensor for analysis of food ingredients

A bioluminescent microbial biosensor induced specifically by l-rhamnose was developed and optimized, to monitor food processes where pectin is involved. The biosensor was developed by fusing the Escherichia coli promoter P_rhaB to the promoterless luxCDABE genes of Vibrio fischeri within plasmid pUCD615. Escherichia coli carrying this construct responded positively to l-rhamnose, but, because of low reporter signal, the enhancement of bioluminescence levels was required. The response was improved by promoter region length manipulation and resulted in 86-fold increase in response ratio. The specificity of the biosensor (80 times higher ratio response to l-rhamnose compared to other carbohydrates) and quantitative response to the target analyte were confirmed. The potential of this biosensor to monitor the hydrolysis of various types of pectin was demonstrated and a calibration curve was acquired. Our study presents a novel l-rhamnose-inducible microbial biosensor as a fast and economical alternative to chemical methods of pectin analysis.     

Bacterial reporter strains for d-xylose content analysis in arabinoxylans

A pair of ice nucleation and fluorescence reporter strains induced specifically by d-xylose were developed and optimized, to monitor d-xylose content in hydrolyzed arabinoxylans samples. Reporter strains were developed by fusing the Escherichia coli xylose isomerase promoter P _xylA to the promoterless native inaZ gene of Pseudomonas syringae and synthetic gfp gene of Aequorea victoria and transferring final constructs to E. coli DH5α. Because of relatively low initial response toward the target analyte, signal improvement by promoter region length adjustment has been implemented. In both cases, this approach proved to be successful, although the manner in which reporter strains have responded to it was dissimilar. The specificity and quantitative response of obtained reporter strains have been confirmed, and the response ranges for NIxylA100 and gfpxylA300 have been established as 9 × 10^?6–9 × 10^?1 g l^?1 and 9 × 10^?3–9 g l^?1, respectively. The performance of developed reporter strains has been assessed in comparison with high-performance anion-exchange chromatography and demonstrated 15–18 % difference between data obtained with reporter strains and chromatography analysis, which for microbial sensors is an acceptable dissimilarity. The developed microbial reporter strains present an alternative for other analytical methods used for monosaccharide quantification and enable quantification of d-xylose in arabinoxylans samples.     

A Validated RP-HPLC-DAD Method for the Determination of l-Theanine in Tea

l-Theanine, an amino acid occurring in the leaves of the tea plant (Camellia sinensis) possesses several beneficial pharmacologic effects. Its effects on the central nervous system are most widely studied, in addition, other activities (potential anticancer, cardiovascular) also stress the significance of this compound. However, analytical methods for the quantification of l-theanine in tea based on the application of HPLC are scarce in the literature. Considering the chemical characteristics of the molecule (high polarity, lack of chromophore group), derivatization and the application of special stationary phases have been preferred for reliable analysis. Here, we report the development and validation of an eligible RP-HPLC-DAD method without the need of pre- or post-column derivatization and using a buffer-free mobile phase (chromatographic separation of l-theanine with water, eluting other compounds with water–acetonitrile gradient) for the determination of l-theanine. The elaborated method provides quick (analysis time 15 mins) and reliable (mean intraday precision 0.94 RSD%, interday precision 0.48 %, recovery >96.1 %, LOQ?=?0.38 mg theanine/g dry tea leaf weight) quantification of this amino acid in aqueous tea extracts.     

A novel α-l-rhamnosidase with potential applications in citrus juice industry and in winemaking

The production of monoglycosylated flavonoids by α-l-rhamnosidases (EC 3.2.1.40) is an interesting development in biocatalysis. Applications of rhamnosidases in industry include removal of bitterness caused by naringin from citrus juices. In the present work, a psychrotolerant bacterial strain with α-l-rhamnosidase activity was isolated. The α-l-rhamnosidase was found to be able to degrade naringin and was purified and characterized. The α-l-rhamnosidase from Brevundimonas sp. Ci19 was able to release both rhamnose and prunin from naringin. The enzyme was partially purified with a performance of 2.7-fold purification. The α-l-rhamnosidase showed an optimum pH between 6.00 and 7.00 with substantial residual activity at pH 5.00 (85.3 %). The optimum temperature was between 20 and 37 °C. The enzyme showed activation in the presence of Ca²⁺ and Cd²⁺ ions and at a high ethanol concentration level (10 % v/v). Activity was found for β-d-glucosidase (EC 3.2.1.21) in the partially purified extract, but it was inactive in the acid pH region. This result indicates the potential for inactivation of β-d-glucosidase along with the high level of α-l-rhamnosidase activity necessary for the production of flavonoid glycosides. The α-l-rhamnosidase from Brevundimonas sp. Ci19 showed interesting properties for potential use not only in the citrus juice industry but also in winemaking.     

Reduction of Acrylamide Formation in Sweet Bread with l-Asparaginase Treatment

Acrylamide, 2-propenamide, has the chemical formula $ \mathrm{CH}2=\mathrm{CH}-\mathrm{CO}-\mathrm{NH}2 $ . It is produced at elevated levels in high temperature fried and baked foods. It has adverse effects on human health and is proven to be neurotoxic, genotoxic, carcinogenic, and toxic to reproductive system. The aim of this paper was to reduce acrylamide formation in bakery products such as sweet bread by enzyme treatment. l-Asparaginase produced from Cladosporium sp. was treated to wheat-based dough at different concentrations (50–300 U). There was no change in the rheological properties of wheat flour and physico-sensory characteristics of bread with l-asparaginase treatment. Moisture, sugars, l-asparagine, acrylamide, and some indicators of Millard reaction (hydroxymethylfurfural (HMF), color, browning) were estimated. With increase in l-asparaginase level the acrylamide formation was reduced. At 300 U, there was 97 % and 73 % reduction of acrylamide formation in the crust and crumb regions of bread, respectively. HMF, a common intermediate product in the Maillard reaction and a genotoxic compound via 5-sulfoxymethylfurfural, also decreased in l-asparaginase-treated bread samples. These results indicated the potential of l-asparaginase enzyme for industrial and domestic applications in reducing harmful Maillard reaction compounds.     

Evaluation of Optimal Conditions for Determination of Low Tin Content in Fresh and Canned Tomato Samples Using Hydride Generation Inductively Coupled Plasma Optical Emission Spectrometry

We propose a procedure for the determination of tin in tomatoes after wet digestion of the samples in HNO₃ using hydride generation inductively coupled plasma optical emission spectrometry. The effect of acidity from HCl and HNO₃ (0.01–4.0 mol L^-1) as well as the presence of l-cysteine on the efficiency of SnH₄ generation by reaction with the NaBH₄ reductant was investigated. Optimal conditions were evaluated in terms of precision, accuracy (by recovery test) and limit of detection. The best results were obtained with l-cysteine and nitric acid for sample acidificitation. Addition of 1 % (m/v) l-cysteine enhanced the Sn signal and expanded the optimal range of HNO₃ concentrations towards higher values; hence, strict control of the acidity could be avoided, up to 1.0 mol L^-1. Under the optimized conditions, a detection limit of 1.2 ng mL^-1 was achieved. The applicability of the proposed method was demonstrated by the determination of low tin contents in digests of fresh and canned tomato samples with satisfactory results.     

l(+)-Lactic acid production from furfural residues and corn kernels with treated yeast as nutrients

In this study, Streptococcus thermophilus and Lactobacillus bulgaricus were used to produce l(+)-lactic acid by simultaneous saccharification and fermentation (SSF). The use of hydrolyzed yeast as cheap nutrients and mixtures of cellulosic materials and starchy materials as carbon source for l(+)-LA production was evaluated. Heat treatment (121 °C) was proven to be an effective method to improve the performances of yeast as nutrients for the fermentations using different carbon sources. The addition of yeast hydrolyzate obviously lowered the surface tension of medium and improved enzyme hydrolysis of furfural residue (FR) as the concentration was beyond 10 g/L. Carbon–nitrogen ratio, substrates composition, substrates feeding rate and enzyme-feeding strategy will affect the productivity of l(+)-LA production from mixed substrates. SSF of FR and corn saccharification liquid tends to obtain good yields, when the total WIS content is in 10 % and carbon–nitrogen ratio is about 30. This study provides an encouraging means of producing l(+)-LA from lignocellulosic resource and starchy resource, which could optimize the use of raw materials.     

HPLC Determination of Lactulose in Heat Treated Milk

Lactulose (4-O-β-d-galactopyranosyl-d-fructose), formed from lactose (4-O-β-d-galactopyranosyl-d-glucose) by the Lobry de Bruyn–Alberda van Ekenstein rearrangement during severe heat treatments, is considered a useful indicator of heat-induced modifications in milk. Its chromatographic determination in milk is particularly troublesome due to the concomitant presence of a lactose amount two orders of magnitude larger than the lactulose amount and to a similar retention time of the two compounds. In this work, four HPLC methods have been compared with the aim to develop a more accurate analytical procedure to determine lactulose in milk, together with lactose. The developed method is based on a Carrez precipitation followed by a HPLC separation on two in-series amino-based columns, using CH₃CN:H₂O 75:25 (v:v) as the mobile phase at 1 ml/min flow rate and a refractive index as the detector. The linearity test for the quantitation of lactulose has been carried out over the range 0.060–1.006 mg/ml, the limit of detection, is 0.013 mg/ml (256 ng injected) and the limit of quantitation is 0.028 mg/ml (556 ng injected). The proposed method, simple, cheap and time-saving, allows an accurate lactulose–lactose separation, with conventional HPLC equipment.     

Correlations of carbon isotope ratios of wine ingredients for the improved detection of adulterations I. Organic acids and ethanol

The δ¹³C-values of organic acids and their correlations to those of the sugar and ethanol, respectively, from 57 EU data bank wines of the Rheinpfalz area (years 1991?–?1993) and from some of their corresponding musts have been determined. In addition to the well established difference between fermented sugar and ethanol (Δδ¹³C = –1.7±0.2‰), a new constant correlation was found in wine for ethanol and citric acid (Δδ¹³C = +2.4±0.4‰). From this result a fixed δ-value difference for citric acid in wine to the fermented sugar of +0.7±0.6‰ can be deduced. The δ¹³C-values of L-malic acid and L-tartaric acid in must were not altered by the alcoholic fermentation; they should therefore directly provide access to the δ¹³C-value of the natural sugar in must. However, in non-adulterated wines the expected δ¹³C-value differences between these acids and ethanol showed unsatisfactory correlation coefficients. For L-malate this is attributed to the secondary (partial) degradation of this acid by the malolactic fermentation; a corresponding correction is envisaged in order to make L-malate available as an internal standard. As a reason for the unsatisfactory correlation between L-tartaric acid and ethanol, it is supposed that the time of its maximum biosynthesis period does not coincide with that of glucose in the grape ripening period.     

Hydrolysis of flatulence causing oligosaccharides by α-d-galactosidase of a probiotic Lactobacillus plantarum MTCC 5422 in selected legume flours and elaboration of probiotic attributes in soy-based fermented product

In the background of increasing research interest in food fermentations involving beneficial culture, the focus present study was to evaluate the ability of a single desirable bacterial culture to impart multiple benefits to the product being prepared therein. The intracellular α-d-galactosidase elaborated by Lactobacillus plantarum MTCC 5422 in formulated soy whey broth for Lactobacillus on partial purification exhibited an activity of 1.2 U/mL with specific activity of 12.0 U/mg and a threefold purification. The partially purified enzyme had a molecular mass of 84 kDa, pH and temperature optima of 5.8 and 45 °C, respectively, and enzyme stability of 30 min at 55 °C. Selected legume flours treated (2 h at 45 °C) with partially purified α-d-galactosidase resulted in lowering raffinose and stachyose levels in the range of 50–74 and 66–85 %, respectively. Soy-based fermented probiotic curd prepared with L. plantarum MTCC 5422 from α-d-galactosidase treated soy protein isolate showed almost 99 % decrease in levels of raffinose and stachyose. In the final product, the probiotic culture of L. plantarum MTCC 5422 reached viable population of 9.4 log₁₀ CFU/g and elaborated short chain fatty acids such as acetic, butyric and propionic acids in appreciable quantity. The product was devoid of any contaminating foodborne pathogenic bacterial species. At the same time, the product had appropriate levels of pH, acidity and antibacterial activity against Bacillus cereus. The innovative approach was the lowering of raffinose and stachyose by α-d-galactosidase of L. plantarum MTCC 5422 and elaboration of probiotic attributes by the same culture in the final product.     

Influence of various factors on formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) in a solid-state model system of Maillard reaction

The influences of various factors on the formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) were investigated in a solid-state Maillard reaction system. Thermogravimetric analysis (TG) and differential scanning calorimetry analysis (DSC) showed that the solid-state Maillard reaction mainly occurred at around 150 °C. The DDMP formation from reducing sugars (glucose or fructose) and several selected amino acids (l-proline, l-alanine, l-asparagine, l-threonine, l-tyrosine and l-lysine) was compared. Proline was found to have special activity in DDMP formation when reacted with glucose because of its special catalytic action. The influences of reactant ratios, oxygen, reaction temperature and reaction time on DDMP formation from the reaction of proline with glucose were studied. Reactant molar ratios of glucose to proline played important impact on DDMP formation, and the formation of DDMP was improved when the ratio was higher than 1:1 and achieved the highest level at 2:1. Oxygen had no obvious effects on DDMP formation. The effects of reaction time and temperature were investigated together and revealed the information on the highest yield and minimum temperature for DDMP formation. Furthermore, the kinetics of DDMP formation was studied, and DDMP formation in the initial stage was shown to follow an apparent first-order reaction with an activation energy of 68.8 kJ/mol.     

Isolation, purification and determination of some biochemical properties of β-glucosidase from Muscat of Bornova grape

This research was undertaken to determine biochemical properties of β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) isolated from Muscat of Bornova grape. The optimum pH for β-glucosidase activity was found to be 5.0, and the enzyme showed high activity over a broad pH range of 4.5–6.0. However, due to low activity at pH 3.0, the enzyme is expected to exhibit only a fraction of the maximum activity during grape juice fermentation due to low pH of grape juice. As the temperature increased from 30 to 55 °C, the activity increased, too, the maximum activity occurring at 55 °C which implies that the enzyme is expected to exhibit a low activity at grape juice fermentation. According to thermal inactivation studies, k _D values increased as the temperature increased, whereas half-life and D values decreased. Energy of activation (E _a) and Z values were found to be 120.99 kj mol^?1 (r ² = 0.9776) and 18.08 °C (r ² = 0.9750), respectively. d-glucose and ethyl alcohol inhibited the enzyme at varying degrees depending on the concentration.     

A maltose,L-rhamnose sensor based on porous Cu foam and electrochemical amperometric i-t scanning method

A maltose, L-rhamnose sensor based on porous Cu foam and electrochemical techniques was investigated in this paper. Cu foam material was prepared and characterized by scanning electron microscopy (SEM). The electro-oxidation reaction process of sweeteners occurred on Cu foam electrode was evaluated by cyclic voltammetry (CV) scanning. At an applied potential of 0.5 V, the linear range for maltose is 0.18–3.47 mM with sensitivity of 1.0492 mA cm^?2 mM^?1. The limit of detection (LOD) was 15.86 μM (S/N?=?3). The linear range for maltose is 0.18–3.47 mM with sensitivity of 0.6881 mA cm^?2 mM^?1. The LOD was 24.18 μM (S/N?=?3). Compared with Cu sheet electrode, Cu foam electrode showed higher current response towards maltose and L-rhamnose, leading to enhanced electrocatalytic activity, higher sensitivity, and lower LOD. Sweetener qualitative discrimination was carried out by stochastic resonance (SR) signal-to-noise ratio (SNR) spectrum eigen peak located noise intensities.     

Preparation of Monodisperse Food-Grade Oleuropein-Loaded W/O/W Emulsions Using Microchannel Emulsification and Evaluation of Their Storage Stability

W/O/W emulsion is an emerging system in developing new functional and low-calorie food products. The aim of this study is to produce food-grade monodisperse water-in-oil-in-water (W/O/W) emulsions loaded with a hydrophilic bioactive oleuropein. W/O/W emulsions were prepared via high-pressure homogenization and subsequent microchannel (MC) emulsification. The internal aqueous phase was a 5-mM sodium phosphate buffer containing d(+)-glucose (5 wt.%) and oleuropein (0.1–0.7 wt.%). The oil phase consisted of soybean oil and tetraglycerin monolaurate condensed ricinoleic acid esters (TGCR; 3–8 wt.%). The external aqueous phase was a 5-mM sodium phosphate buffer containing d(+)-glucose (5 wt.%) and decaglycerol monolaurate (1 wt.%). Oleuropein-loaded submicron W/O emulsions with average droplet diameters as small as 0.15 μm and monomodal droplet size distributions were prepared by high-pressure homogenization when applying high TGCR concentrations of 5–8 wt.% and low oleuropein concentrations of 0.1–0.3 wt.%. Monodisperse oleuropein-loaded W/O/W emulsions with average W/O droplet diameters of around 27 μm and coefficients of variation of below 5 % were successfully prepared when using a silicon MC array plate with wide channels of 5-μm depth and 18-μm width. The monodisperse W/O/W emulsions prepared at high TGCR concentrations and low oleuropein concentrations were the most stable during 40 days of storage. The adsorption behavior of oleuropein at the internal aqueous–oil interface was relevant to W/O/W emulsions microstructure and stability. The results are believed to provide useful information for successfully preparing stable monodisperse W/O/W emulsions loaded with hydrophilic functional compounds. The surface activity of the loaded material seems to be a key parameter in optimizing the formulation of W/O/W food emulsion.     

First extraction of polyphenol oxidase from edible desert truffle (Terfezia leonis Tul.) and its thermal behavior

Polyphenol oxidase (PPO) is a common enzyme with large applications in food processing and analysis, especially based on their monophenolase activity. In this context, extraction and surfactant-mediated activation of PPO from desert truffle Terfezia leonis Tul. were successfully achieved. In the presence of l-tyrosine, the cresolase activity was optimal in the pH 5–6 domain and in the 35–45 °C temperature range. In the presence of pyrocatechol, the catecholase activity was optimal at neutral pH and 30 °C. Kinetics studies revealed higher affinity of PPO for l-tyrosine than for pyrocatechol. Both enzyme–substrate complexes were structurally robust, and their thermosensitivity was mainly related to entropy changes. These properties may reflect the adaptation to desert conditions where T. leonis grows and should be useful for the development of enzymatic catalysts and sensors.     

Freezing Characteristics and Storage Stability of Broccoli (Brassica oleracea L. var. botrytis L.) Under Osmodehydrofreezing and Ultrasound-Assisted Osmodehydrofreezing Treatments

Freezing characteristics (freezing time, latent heat of fusion of ice, and freezable water content) and quality parameters (drip loss, color, firmness, and l-ascorbic acid content) of broccoli (osmodehydrofrozen and ultrasound-assisted osmodehydrofrozen) during frozen storage were investigated. Freezing time, latent heat of fusion of ice, and freezable water content of osmodehydrated samples decreased significantly compared to samples which were not under osmotic dehydration. The changes of drip loss, color, firmness, and l-ascorbic acid content of osmodehydrofrozen and ultrasound-assisted osmodehydrofrozen broccoli during frozen storage were inhibited markedly compared to samples which were not submitted to osmotic dehydration before freezing. Compared to osmotic dehydration, the ultrasound-assisted osmotic dehydration shortened the needed dehydration time and better preserved the firmness and l-ascorbic acid content after osmotic dehydration pretreatment. In addition, the ultrasound-assisted osmotic dehydration minimized the drip loss and loss of l-ascorbic acid content and better maintained the color and firmness when stored at ?25 °C for 6 months. These findings indicate that it is promising to apply ultrasound-assisted osmodehydrofreezing in freezing and frozen storage of food.     

Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a

The aim of this study was to purify a malolactic enzyme (MLE) from Oenococcus oeni (O. oeni) strain and determine its properties in detail. O. oeni SD-2a was cultivated in the ATB broth supplemented with 7 g/L l-malic acid for harvesting the cells. After harvest, the cells were washed and disrupted for purification of MLE. MLE was purified from the supernatant of the disrupted cells through protamine sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purified MLE was identified using mass spectrometry. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme with a nominal molecular mass of 59 kDa and a theoretical pI of 4.76 exhibited a maximum enzyme activity at 35 °C and pH 6.0, which retained over 50 % of its initial activity in the presence of 14 % (v/v) ethanol. Mn²⁺ was proven to be the most effective divalent cation to promote enzyme activity. Under the conditions of temperature 30 °C and pH 6.0, the K _m and V _max of MLE on l-malic acid were 12.5 × 10^?3 M and 43.86 μmol/(min × mg), respectively. Moreover, the purified enzyme exhibited a higher stability with 0.1 M NaCl in addition and had a half-life of 30 days at 4 °C.     

Microwave treatment of dietary gelatin does not generate cis-4-hydroxy-l-proline, an inhibitor of collagen biosynthesis

Aqueous solutions (5?g/100?ml) of commercial preparations of (a) an enzymatic partial hydrolysate of gelatin and (b) type A gelatin were subjected to threefold heating to boiling in a domestic microwave oven at 750?W and to conventional heating. Then samples were totally hydrolyzed (6?M hydrochloric acid, 110??°C, 24?h) and investigated for the presence of eight possible stereoisomers of 3- and 4-hydroxyproline (Hyp) using capillary gas chromatography. Amino acids were analyzed as N(O)-trifluoroacetyl 2-propyl esters on Chirasil-l-Val and detected by selected ion monitoring mass spectrometry. Blanks of (a) and (b) were analyzed in parallel. Relative amounts of 5.0±0.2% cis-4-d-Hyp were generated from native trans-4-l-Hyp as a result of total hydrolysis in all samples and independent of previous treatment. Notably, neither cis-3-l-Hyp nor cis-4-l-Hyp could be detected in either of the gelatin samples. Thus a report on the generation of antifibrotic and therefore potentially hazardous cis-3-l-Hyp and cis-4-l-Hyp from protein-bonded native trans-3-l-Hyp and trans-4-l-Hyp on microwave heating of infant formulae could not be confirmed.