Digestive and metabolic bioavailability in healthy humans of 15N-labeled rapeseed and flaxseed protein incorporated in biscuits.

BACKGROUND: In the search to diversify protein sources for humans, oilseeds are good candidates due to the high protein content of their coproducts after oil extraction. Among them, rapeseed presents a well-balanced amino acid (AA) profile. Flaxseed is an emerging source but the nutritional value of its protein is not yet documented. OBJECTIVES: This study aimed to determine the nitrogen (N) and AA bioavailability of these protein sources. METHODS: Nineteen healthy volunteers were intubated with a naso-ileal tube. They ingested 156 g biscuits containing intrinsically labeled 15N rapeseed (n = 10) or flaxseed (n = 9) protein over a 4-h period. Ileal digesta, blood, and urine were sampled over 8 h after the first meal ingestion. N and 15N enrichment and AAs were measured to determine digestive and deamination losses. Ileal digestibility, the digestible indispensable AA score (DIAAS) and net postprandial protein utilization (NPPU) were calculated. RESULTS: Real ileal digestibility was 80.7 ± 6.5% for rapeseed protein and 92.2 ± 2.0% for flaxseed protein (P = 0.0002). Mean indispensable AA (IAA) digestibility reached 84.1 ± 6.9% and 93.3 ± 6.7% for rapeseed and flaxseed, respectively, lysine being the lowest digestible IAA for both sources. Despite moderate digestibility, the DIAAS was 1.1 for rapeseed but only 0.6 for flaxseed due to lysine insufficiency. Deamination losses accounted for 20.0 ± 6.5% of dietary N for flaxseed and 11.0 ± 2.8% for rapeseed (P = 0.002). The NPPU did not differ between the protein sources, with 71.3 ± 6.5% for flaxseed and 69.7 ± 7.6% for rapeseed. CONCLUSIONS: Despite good digestibility, flaxseed protein cooked in biscuits was penalized by both lysine insufficiency and poor lysine digestibility that decreased its DIAAS and increased deamination. By contrast, rapeseed was moderately digestible but presented no limiting IAA, resulting in an excellent DIAAS and low deamination. This study was registered at clinicaltrials.gov as NCT04024605.

Improving the in vitro digestibility of rapeseed albumins resistant to gastrointestinal proteolysis while preserving the functional properties using enzymatic hydrolysis.

Study of in vitro digestibility of high-quality isolate of rapeseed albumins (RA) was carried out in this work, using Size-Exclusion Chromatography. A poor in vitro digestibility of the RA isolate was highlighted (15%). The aim of this study was therefore to improve the RA in vitro digestibility by enzymatic hydrolysis while preserving its attractive functional properties. Alcalase, Flavourzyme and Prolyve were used to obtain 12 hydrolysates with various degrees of hydrolysis (DH) and compositions. All hydrolysates showed improved digestibility and those with the highest DH showed the best improvements. Techno-functional properties of these hydrolysates were also characterized. The poor emulsion capacity of initial RA was improved and results showed that extent proteolysis can be a good way to improve both digestibility and functional properties. Moreover, optimal conditions for RA proteolysis were identified to produce with Flavourzyme a partial hydrolysate (still containing 50% intact RA) that is both digestible and functional.

Combined Effect of Extraction and Purification Conditions on Yield, Composition and Functional and Structural Properties of Lupin Proteins.

Lupin meal presents great potential as an alternative plant-based source of proteins for human nutrition. In the present work, different conditions of extraction and purification were evaluated for production of lupin protein isolates. The results showed that the protein extraction yield was comparable at acidic and conventionally used alkaline extraction pH (37% vs. 40-45%, respectively). Proteins extracted were principally composed of globulins. The ionic strength negatively impacted the protein extractability at pH 2, whereas no significant differences were observed between extractions at 20 to 50 °C. The selected extraction conditions (pH 2 and 7) combined with purification by isoelectric precipitation or ultrafiltration process generated the isolate-grade products. Interestingly, further characterization revealed a partial denaturation of proteins extracted at pH 2 resulting in loss of protein solubility at pH 6 and 7 (10-50%), modifications in secondary structure, lower thermal stability, and formation of protein aggregates. However, foaming and emulsifying properties were generally similar for almost all lupin isolates. Further investigation might be of interest with regard to the extraction behaviours and structural and functional properties of specific lupin protein fractions.

The True Amino Acid Digestibility of 15N-Labelled Sunflower Biscuits Determined with Ileal Balance and Dual Isotope Methods in Healthy Humans.

BACKGROUND: Sunflower is a promising protein source but data on amino acid (AA) digestibility are lacking in humans. Classically, the determination of AA digestibility requires ileal digesta sampling. The dual isotope method is minimally invasive but has not been compared to the conventional approach. OBJECTIVES: This study aimed to determine the true ileal digestibility of sunflower AAs in healthy volunteers who ate biscuits containing 15nitrogen (N) protein isolate, in comparison with the dual isotope method. METHODS: Twelve healthy volunteers (men and women; 40.4 ± 10.5 years old; BMI, 23.7 ± 2.9 kg/m2) were equipped with a naso-ileal tube. For 4 hours, they consumed 9 repeated meals comprising 15N-sunflower protein biscuits together with 13carbon (C)-AAs, carried either in chocolate (SUN + Ch; n = 7) or apple puree (SUN + P; n = 5). Ileal digesta and blood were sampled throughout 8 hours after ingestion of the first meal. The 15N and 13C AA enrichments were measured in digesta to determine ileal digestibility directly and in plasma to determine lysine and threonine digestibility using the dual isotope method. Differences between methods and between vector groups were analyzed using paired and unpaired t-tests, respectively. RESULTS: The ileal digestibility of sunflower indispensable AAs (IAA) was 89% ± 5.3%, with threonine and lysine having the lowest digestibility. In the SUN + Ch meal, IAA digestibility was 3% below that of SUN + P (P < 0.05). The mean free 13C-AA ileal digestibility was 98.1% ± 0.9%. No matter which matrix was used to carry 13C-AAs, plasma 15N and 13C-AA kinetics displayed a 1-hour offset. Digestibility obtained with the dual isotope method (70.4% ± 6.0% for threonine and 75.9% ± 22.3% for lysine) was below the target values. CONCLUSIONS: The ileal digestibility of IAAs from a sunflower isolate incorporated in a biscuit was close to 90% in healthy adults. Under our experimental conditions, the dual isotope method provided lower values than the usual method. Further protocol developments are needed to validate the equivalence between both methods. This trial was registered at clinicaltrials.gov as NCT04024605.

Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8.

SCOPE: Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model. METHODS AND RESULTS: The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity. CONCLUSIONS: SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.

A Rational Approach for the Production of Highly Soluble and Functional Sunflower Protein Hydrolysates.

Exploitation of plant proteins as an alternative to animal proteins currently presents an important challenge for food industries. In this contribution, total sunflower protein isolate from cold press meal was used as a starting material for the generation of highly soluble and functional hydrolysates that could be used in various food formulations. To do this, a rational and complete approach of controlled hydrolysis was implemented using the individual Alcalase and Prolyve enzymes. The method of stopping the hydrolysis reaction was also evaluated. The influence of operating conditions on hydrolysis kinetics and enzymatic mechanism was studied to identify the appropriate hydrolysis conditions. The gain of the solubility was then analyzed and compared to that of the initial proteins. Finally, the emulsifying and foaming properties (capacities and stabilities) of the resulting hydrolysates were also assessed. As a result, controlled enzymatic proteolysis significantly improved the sunflower protein solubility at neutral pH (twofold increase) and generated highly soluble hydrolysates. The limited proteolysis also maintained the good foam capacities and allowed an improvement in the initial foam stabilities and emulsifying capacities and stabilities of sunflower proteins. This contribution can greatly increase the value of sunflower meal and help in the development of sunflower protein products in the future.

Multi-objective optimization of solid/liquid extraction of total sunflower proteins from cold press meal.

The impact of pH (6-9) and NaCl concentration (0-0.5 mol.L-1) on sunflower protein extraction was studied through design of experiments. The considered criteria were protein extraction yield (total proteins, helianthinin and albumins), chlorogenic acids covalently bound to proteins, and free chlorogenic acid concentration in the aqueous extract. Statistical analysis showed that the obtained by design of experiments the polynomial models of each extraction criteria were reliable for predicting the responses. They were employed in an original multi-objective optimization methodology. The optimal conditions revealed to be pH 7.3/0.3 mol.L-1 NaCl yielded 46.83% and 59.16% of total protein and albumin extraction yield, 1.730 and 1.998 mg.g-1 of chlorogenic acids covalently bound to helianthinin and albumins in aqueous extract, respectively. The sunflower protein isolate obtained after extraction in this condition had good solubility (40-80% at pH 5-8), functional properties (foaming and emulsifying) and a satisfying color.

Identification of the bacteria and their metabolic activities associated with the microbial spoilage of custard cream desserts.

The famous French dessert "ile flottante" consists of a sweet egg white foam floating on a vanilla custard cream, which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a key concern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream desserts manufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg.

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

A new SE-HPLC method for simultaneous quantification of proteins and main phenolic compounds from sunflower meal aqueous extracts.

The aim of this research was to develop a method for simultaneous quantification of proteins and main polyphenolic compounds extracted from oleaginous meal by aqueous media. Size exclusion chromatography with a Biosep column (exclusion range from 1 to 300 kDa) and acetonitrile/water/formic acid (10:89.9:0.1 v/v) eluent at 0.6 mL min-1 yielded the most efficient separation of sunflower proteins and chlorogenic acid monoisomers (3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid). After a study of the stability of the extract components, the incorporation of a stabilization buffer (0.5 mol L-1 tris(hydroxymethyl)aminomethane-hydrochloric acid/1.0 mol L-1 sodium chloride at pH 7) was proposed to avoid polyphenol-protein interactions and/or isomeric transformation. The use of 214 nm as the wavelength for protein quantification was also included to minimize the effect of interference from polyphenol-protein interactions on the quantification. Under the used experimental conditions, the protein and chlorogenic acid monoisomer signals remained stable during 300 min at 20 °C (95-125% of the starting value). The developed method was validated and parameters such as specificity, sensitivity, precision, and accuracy were determined. The results from size exclusion chromatography correlated well with the results of protein determination by the reference Kjeldahl method. The proposed method was successfully applied for rapeseed extract analysis making simultaneous quantification of proteins and major rapeseed polyphenols (sinapine and sinapic acid) possible. Graphical abstract.

Simultaneous quantification of the degree of hydrolysis, protein conversion rate and mean molar weight of peptides released in the course of enzymatic proteolysis.

This paper describes an original analytical methodology for a simultaneous measurement of the protein conversion rate, the mean molar weight of peptide and the degree of hydrolysis in the course of proteolysis by Size-Exclusion High-Performance Liquid Chromatography. Peak area of dead volume eluents reflects the non-converted protein. The protein conversion rate is thus determined by comparing the area at a given time to the initial area. The peptide signal allows determining the peptide molar weight distribution and degree of hydrolysis of hydrolysates. As a first step, the approach was tested on the hydrolysis of bovine serum albumin, lysozyme and rapeseed albumin by Alcalase 2.4L. Values of degree of hydrolysis were also determined by TNBS and pH-stat methods. Most of the hydrolysate obtained showed relative differences < 20% with the reference methods. The method was also adapted to fit the TNBS assay. 39 experimental validation tests were analyzed by size-exclusion chromatography, TNBS and pH stat methods. 90% of the validation data show non-significant differences between the degree of hydrolysis predicted and the degree of hydrolysis measured by TNBS method. Hence, the proposed methodology can be efficient to study the process of enzymatic proteolysis while minimizing time and quantity of sample assay required.