Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein-protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops.

BACKGROUND: Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. RESULTS: In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. CONCLUSIONS: The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.

Comparative assessment of physico-chemical characteristics and fibril formation capacity of thermostable carp scales collagen.

Collagen and collagen fibers have been widely documented as a potential and competitive biomaterial for medical applications. However, the searches for safe and realistic new collagen sources are still underway. Currently, fishery by-products (scales), a promising collagen source are usually discarded. In the present study, in vitro fibril-forming ability of the extracted fish scale collagen is reported. The aim of the investigation was to evaluate the concomitant comparison of fibril-forming abilities and characteristics of acid and pepsin soluble collagens from the scales of Indian major carp catla (Catla catla) and rohu (Labeo rohita). The extracted collagens were characterized as type I, with a total yield of 2.80-4.11% (w/w). Denaturation temperature determined for all collagens were between 35.9 and 37.7°C. All collagens exhibited high solubility in acidic pH and low NaCl concentrations. SEM clarified the lyophilized collagens and their fibril-forming capacity. Amino acid content and radical scavenging efficacy were also analyzed for the extracted collagen. The results revealed that extracted scale collagen from a renewable biological source could be used as biomaterials in various sectors. It might be suitable for preparing collagen gel for biomedical devices or as a scaffold for cell culture because of its high stability and fibril formation capacity.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Isolation and characterization of pepsin-solubilized collagen from the integument of sea cucumber (Stichopus vastus).

BACKGROUND: Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the purified collagen was of type I, consisting of three α1 chains of approximately 122 kDa each. The peptide map of PSC digested by V8 protease was different from that of calf skin type I collagen. Fourier transform infrared spectroscopy revealed that the triple helical structure was well preserved in isolated collagen. The denaturation temperature of PSC was 21.23 °C and showed good gel-forming capability at pH 6.5 and 300 mmol L⁻¹ NaCl. CONCLUSION: It is inferred that the collagen isolated from S. vastus integument has potential for use as an alternative to land-based mammalian collagen in food, nutraceuticals and pharmaceutical industries.

Biofortification of rice with zinc: assessment of the relative bioavailability of zinc in a Caco-2 cell model and suckling rat pups.

Staple foods, such as rice, can now be enriched in micronutrients through conventional breeding (i.e., biofortification) to enhance dietary intake of vulnerable populations. The objectives of this study were (1) to establish a rapid, high capacity Caco-2 cell model to determine the relative bioavailability of zinc (Zn) from samples of staple food breeding lines for potential use as a guideline for selection/breeding and (2) to determine the relative bioavailability of Zn from conventional rice varieties and one Zn-biofortified type. Polished or undermilled, parboiled rice samples were digested in vitro with pepsin and pH adjustment, and by pancreatic enzymes. Zn uptake from digested samples was measured in Caco-2 cells in culture. A previously validated rat pup model was also used to assess Zn absorption in vivo, using gastric intubation and (65)Zn labeling. Pups were killed after 6 h, and radioactivity in tissues and in small intestine perfusate and cecum-colon contents was used to measure Zn bioavailability. A biofortified rice variety contained substantially more Zn than conventional varieties, with no change in phytate content. Absorbed Zn (µg/g rice) was significantly higher from the new variety in both the in vitro Caco-2 cell model (2.1-fold) and the rat pup model (2.0-fold). Results from the two models were highly correlated, particularly for the polished samples. Biofortification of rice with Zn results in significantly increased Zn uptake in both models. Since results from the Caco-2 cell model correlated well with those from rat pups, this cell model is likely to predict results in human populations and can be used for screening purposes.

In vitro assessment of the bioaccessibility of fatty acids and tocopherol from soybean oil body emulsions stabilized with ι-carrageenan.

The present investigation aimed to expand the knowledge of the in vitro bioaccessibility of fatty acids and tocopherol from natural soybean oil body emulsions stabilized with different concentrations of ι-carrageenan. Several physicochemical parameters including proteolysis of the interfacial layer, interfacial composition, and microstructure were evaluated with regard to their impact on the bioaccessibility of fatty acids and tocopherol. Results from simulated human digestion in vitro indicated that the bioaccessibility of total fatty acids and tocopherol decreased (62.7-8.3 and 59.7-19.4%, respectively) with the increasing concentration of ι-carrageenan. During the in vitro digestion procedure, ι-carrageenan affected physicochemical properties of the emulsions, thereby controlling the release of fatty acids and tocopherol. These results suggested that soybean oil body emulsions stabilized with ι-carrageenan could provide natural emulsions in foods that were digested at a relatively slow rate, the important physiological consequence of which might be increasing satiety.

Allergenicity assessment of Allium sativum leaf agglutinin, a potential candidate protein for developing sap sucking insect resistant food crops.

BACKGROUND: Mannose-binding Allium sativum leaf agglutinin (ASAL) is highly antinutritional and toxic to various phloem-feeding hemipteran insects. ASAL has been expressed in a number of agriculturally important crops to develop resistance against those insects. Awareness of the safety aspect of ASAL is absolutely essential for developing ASAL transgenic plants. METHODOLOGY/PRINCIPAL FINDINGS: Following the guidelines framed by the Food and Agriculture Organization/World Health Organization, the source of the gene, its sequence homology with potent allergens, clinical tests on mammalian systems, and the pepsin resistance and thermostability of the protein were considered to address the issue. No significant homology to the ASAL sequence was detected when compared to known allergenic proteins. The ELISA of blood sera collected from known allergy patients also failed to show significant evidence of cross-reactivity. In vitro and in vivo assays both indicated the digestibility of ASAL in the presence of pepsin in a minimum time period. CONCLUSIONS/SIGNIFICANCE: With these experiments, we concluded that ASAL does not possess any apparent features of an allergen. This is the first report regarding the monitoring of the allergenicity of any mannose-binding monocot lectin having insecticidal efficacy against hemipteran insects.

Collagens from the skin of arabesque greenling (Pleurogrammus azonus) solubilized with the aid of acetic acid and pepsin from albacore tuna (Thunnus alalunga) stomach.

BACKGROUND: Due to the low extraction efficiency of collagen from fish skin by the typical acid solubilization process, pepsin has been widely used to aid further extraction of collagen from the residue. The aim of this study was to characterize collagen from the skin of arabesque greenling extracted with the aid of albacore tuna pepsin, in comparison with collagen obtained from the acid solubilization process. RESULTS: Acid-solubilized collagen (ASC) from the skin of arabesque greenling was extracted with acetic acid. Pepsin-solubilized collagen (PSC) was further extracted from the skin residue with the aid of pepsin from albacore tuna. The yields of ASC and PSC were 303 and 140 g kg(-1) (dry weight), respectively. Both collagens contained alpha- and beta-chains as their major components and were characterized as type I collagen. Both collagens contained glycine as a major amino acid and had imino acid content of 157-159 residues per 1000 residues. The degradation induced by lysyl endopeptidase and V8-protease was more pronounced in PSC compared with ASC. Maximal transition temperatures of both collagens were in the range of 15.4-15.7 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between ASC and PSC. Nevertheless, the triple-helical structure of PSC was still predominant. Based on zeta-potential, pI of ASC and PSC was estimated to be 6.31 and 6.38, respectively. CONCLUSION: Isolation of collagens from the skin of arabesque greenling could be achieved by acid or albacore tuna pepsin solubilization. However, there was a slight difference in properties between ASC and PSC.

In vitro assessment of the bioaccessibility of tocopherol and fatty acids from sunflower seed oil bodies.

The in vitro digestibility (proteolytic and lipolytic) and bioaccessibility of nutritionally important compounds (alpha-tocopherol and fatty acids) have been studied for natural sunflower ( Helianthus annuus ) oil body suspensions in comparison to artificial emulsions emulsified with polyoxyethylene-20-sorbitan-monolaurate (Tween 20) or whey protein isolate. Proteolytic digestion of emulsions with pepsin (pH 2) promoted significant increases in mean particle size of the whey protein isolate stabilized emulsion (1.8-2.9 mum) and oil bodies (2.3-22.5 mum) but not the Tween 20 stabilized emulsions. SDS-PAGE of proteolytic digestion products suggested degradation of the stabilizing oleosin protein (ca. 18-21 kDa) in oil bodies. The rate of oil body hydrolysis with lipase was significantly slower than the lipase-catalyzed hydrolysis of the artificial emulsions and exhibited a prolonged lag phase. Results from simulated human digestion in vitro suggested that the mean bioaccessibility of alpha-tocopherol and total fatty acids from oil bodies (0.6 and 8.4%, respectively) was significantly lower than that from the Tween 20 stabilized emulsion (35 and 52%, respectively) and the whey protein isolate stabilized emulsion (17 and 33%, respectively). These in vitro results suggest that oil bodies could provide a natural emulsion in food that is digested at a relatively slow rate, the physiological consequence of which may be increased satiety.

Current codex guidelines for assessment of potential protein allergenicity.

A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduced protein as well as the crop containing such protein with the goal of demonstrating the GM crop is "as-safe-as" non-transgenic crops in the food supply. One of the major issues for GM crops is the assessment of the expressed protein for allergenic potential. Currently, no single factor is recognized as an identifier for protein allergenicity. Therefore, a weight-of-evidence approach, which takes into account a variety of factors and approaches for an overall assessment of allergenic potential, is conducted [Codex Alimentarious Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarious Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity, pp. 47-60]. This assessment is based on what is known about allergens, including the history of exposure and safety of the gene(s) source; protein structure (e.g., amino acid sequence identity to human allergens); stability to pepsin digestion in vitro [Thomas, K. et al., 2004. A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul. Toxicol. Pharmacol. 39, 87-98]; an estimate of exposure of the novel protein(s) to the gastrointestinal tract where absorption occurs (e.g., protein abundance in the crop, processing effects); and when appropriate, specific IgE binding studies or skin prick testing. Additional approaches may be considered (e.g., animal models; targeted sera screening) as the science evolves; however, such approaches have not been thoroughly evaluated or validated for predicting protein allergenicity.

Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods.

RATIONALE: Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity. METHODS: Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS-PAGE. Residual stained protein bands were measured by image analysis. RESULTS: The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins. CONCLUSIONS: Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.

Differences in allergenic potential of food extracts following oral exposure in mice reflect differences in digestibility: potential approaches to safety assessment.

An animal model for food allergy is needed to assess genetically modified food crops for potential allergenicity. The ideal model must produce allergic antibody (IgE) to proteins differentially according to known allergenicity before being used to accurately identify potential allergens among novel proteins. The oral route is the most relevant for exposure to food antigens, and a protein's stability to digestion is a current risk assessment tool based on this natural route. However, normal laboratory animals do not mount allergic responses to proteins administered orally due to oral tolerance, an immunologic mechanism which specifically suppresses IgE. To circumvent oral tolerance and evoke differential IgE responses to a panel of allergenic and nonallergenic food extracts, female C3H/HeJ mice were exposed subcutaneously or orally with cholera toxin as an adjuvant. All foods elicited IgE by the subcutaneous route. Oral exposure, however, resulted in IgE to allergens (peanut, Brazil nut, and egg white) but not to nonallergens (spinach and turkey), provided that the dose and exposures were limited. Additionally, in vitro digestibility assays demonstrated the presence of digestion-stable proteins in the allergenic food extracts but not in the nonallergenic foods. Our results suggest that the subcutaneous route is inadequate to distinguish allergens from nonallergens, but oral exposure under the appropriate experimental conditions will result in differential allergic responses in accordance with known allergenicity. Moreover, those foods containing digestion-resistant proteins provoke allergic responses in this model, supporting the current use of pepsin resistance in the decision tree for potential allergenicity assessment.

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan.

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.

In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium.

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.

Evaluation of the potential allergenicity of the enzyme microbial transglutaminase using the 2001 FAO/WHO Decision Tree.

All novel proteins must be assessed for their potential allergenicity before they are introduced into the food market. One method to achieve this is the 2001 FAO/WHO Decision Tree recommended for evaluation of proteins from genetically modified organisms (GMOs). It was the aim of this study to investigate the allergenicity of microbial transglutaminase (m-TG) from Streptoverticillium mobaraense. Amino acid sequence similarity to known allergens, pepsin resistance, and detection of protein binding to specific serum immunoglobulin E (IgE) (RAST) have been evaluated as recommended by the decision tree. Allergenicity in the source material was thought unlikely, since no IgE-mediated allergy to any bacteria has been reported. m-TG is fully degraded after 5 min of pepsin treatment. A database search showed that the enzyme has no homology with known allergens, down to a match of six contiguous amino acids, which meets the requirements of the decision tree. However, there is a match at the five contiguous amino acid level to the major codfish allergen Gad c1. The potential cross reactivity between m-TG and Gad c1 was investigated in RAST using sera from 25 documented cod-allergic patients and an extract of raw codfish. No binding between patient IgE and m-TG was observed. It can be concluded that no safety concerns with regard to the allergenic potential of m-TG were identified.

Assessment of the potential allergenicity of ice structuring protein type III HPLC 12 using the FAO/WHO 2001 decision tree for novel foods.

The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein. Therefore, decision trees for evaluation of the risk have been developed, the latest being proposed by WHO/FAO early in 2001. Proteins developed using modern biotechnology and derived from fish are being considered for use in food and other applications, and since allergy to fish is well established, a potential risk from such proteins to susceptible human beings exists. The overall aim of the study was to investigate the potential allergenicity of an Ice Structuring Protein (ISP) originating from an arctic fish (the ocean pout, Macrozoarces americanus) using the newly developed decision tree proposed by FAO/WHO. The methods used were those proposed by FAO/WHO including amino acid sequence analysis for sequence similarity to known allergens, methods for assessing degradability under standardised conditions, assays for detection of specific IgE against the protein (Maxisorb RAST) and histamine release from human basophils. In the present paper we describe the serum screening phase of the study and discuss the overall application of the decision tree to the assessment of the potential allergenicity of ISP Type III. In an accompanying paper [Food Chem. Toxicol. 40 (2002) 965], we detail the specific methodology used for the sequence analysis and assessment of resistance to pepsin-catalysed proteolysis of this protein. The ISP showed no sequence similarity to known allergens nor was it stable to proteolytic degradation using standardised methods. Using sera from 20 patients with a well-documented clinical history of fish allergy, positive in skin prick tests to ocean pout, eel pout and eel were used, positive IgE-binding in vitro to extracts of the same fish was confirmed. The sera also elicited histamine release in vitro in the presence of the same extracts. The ISP was negative in all cases in the same experiments. Using the proposed decision tree, we demonstrated the safety of the ISP to patients already sensitised to fish, as well as to individuals potentially susceptible to producing IgE responses to proteins. Furthermore, the practicability of the new decision tree was confirmed.

Sequence analysis and resistance to pepsin hydrolysis as part of an assessment of the potential allergenicity of ice structuring protein type III HPLC 12.

The recently published WHO/FAO guidelines on the assessment of allergenicity of novel food proteins provide a strategy with which to approach the determination of the potential of novel proteins in foods to be allergens. Key to this strategy are the assessment of sequence similarity to known allergens and the assessment of the resistance to pepsin hydrolysis. Ice structuring proteins (also commonly referred to as anti-freeze or thermal hysteresis proteins) are a group of naturally occurring proteins that bind to ice and structure ice crystal formation. The amino acid sequence of the ice structuring protein (ISP) type III HPLC 12 (ISP type III) was compared in silico with the sequences of known allergens. Secondly, the resistance to pepsin hydrolysis of ISP type III and its glycoconjugates (produced in recombinant baker's yeast) was assessed. The results indicate that ISP type III has no sequence similarity with known allergenic proteins. Both ISP type III and ISP type III glycoconjugates contained within the fermentation product were hydrolysed readily by pepsin (50% loss in <10 min at pH 1.5) to give peptide fragments that were too small to be allergenic or to trigger cross-linking to IgE. In an accompanying study, we demonstrated that IgE from fish-allergic individuals did not bind ISP Type III. Therefore, in accordance with the WHO/FAO strategy, the assessment of ISP type III and ISP type III glycoconjugates by sequence analysis together with lack of resistance to pepsin hydrolysis and the absence of IgE binding supports the conclusion that both are unlikely to present a potential sensitisation hazard.

Assessment of nutritional adequacy of the protein in dog foods by trials on growing rats.

In order to offer methods for assessing the protein quality of dog foods, eight commercial dry-type complete dog foods were analysed by in vitro chemical methods (crude protein, amino acid composition, in vitro pepsin digestibility) and rat growth trial (weight gain = WG, feed efficiency = FE, protein efficiency ratio = PER, net protein ratio = NPR, and net protein utilisation = NPU). Three individual samples were taken from each dog food and their homogenous mixture was used as an ingredient for the test diets fed to six rats per diet. All dog food samples were used as a single protein source in isonitrogenous (10% crude protein) and isoenergetic (ME = 14.9 MJ/kg) complete diets. Rat weight gains showed significant differences among most of the test diets as a consequence of the differences in protein quality of the dog food samples. The protein quality indices of the dog food products showed significant variations. The measured ranges of protein quality indices of the samples were as follows: WG = -5.4-21.4 g; FE = 0-0.25 g/g; PER = 0-2.54 g/g; NPR = 1.06-3.52 g/g; NPU = 19.93-65.93%. The limiting amino acid, the sum of essential amino acids and the chemical scores were calculated from gross amino acid composition. Tryptophan was the first limiting amino acids in all samples but one. Methionine was found to be the second limiting amino acid. The crude protein content of the samples showed no correlation with indices of protein quality, indicating that the declaration of crude protein is not a sufficient information to judge the protein quality of dog foods. The declaration of NPR, or NPU indices may be recommended for labelling the protein quality of premium-type dog foods.