Method for the formulation of a gel-format foodstuff for use as a nutritional foodstuff enriched with peptides and maltodextrins obtained from quinoa flour

Abstract

Disclosed is a process to extract peptides and maltodextrins from quinoa flour for the manufacturing of foodstuff corresponding to a gel for sportspeople consumption during and after physical activity.

Claims

1. A procedure for preparing a peptide and maltodextrin enriched gel foodstuff from quinoa starch, comprising the steps of: a) extracting protein from quinoa flour having a granulometry between 100-300 μm with 30 mL of sodium hydroxide 40 mM per gram of flour under constant stirring and at room temperature, wherein the sodium hydroxide is centrifuged maintaining in a supernatant 0.3-0.4% w/v of soluble protein; b) recovering proteins by isoelectric precipitation and further centrifugation and then performing an enzymatic treatment comprising the performance of two or more sequential enzymatic hydrolysis of the protein concentrate resulting from the isoelectric precipitation and drying the recovered proteins, wherein the first enzymatic hydrolysis of the extracting step a) comprises incubating with alkalase at a temperature higher than room temperature to achieve pH 8.0-8.5, the second enzymatic hydrolysis comprises an enzymatic hydrolysis with protease having a temperature higher than room temperature and pH 7; and the temperature higher than room temperature ranges from 50° C. to 60° C., to obtain an amount of 12.4% of peptides, wherein 55% of the peptides as obtained have a molecular weight (MW) ranging 0.5-1 kDa while 35% of the peptides as obtained have a MW lower than 0.5 kDa; c) extracting maltodextrin from quinoa flour having a granulometry <<100 μm and a solid fraction from precipitate obtained from step a), including starch, by adjusting the solution to a slightly acidic pH and admixing CaCl.sub.2 and performing an enzymatic hydrolysis and obtaining maltodextrins to perform a step of drying after the enzymatic treatment; and d) creating a homogeneous mixture by combining the solution of quinoa flour having ganulometry <<100μm, which is mainly constituted by starch with the soluble protein obtained from the step of extraction a) and maltodextrins obtained from the step of extraction c) adding optionally, colorants, flavors and preservatives to subsequently adding water and heating under constant stirring with the objective of gelatinizing the mixture of starch of quinoa enriched in peptides and maltodextrines and once achieved the desired consistency it is cooled at room temperature.

2. The procedure according to claim 1, wherein in step a), the centrifuging is performed at a temperature of 4° C.

3. The procedure according to claim 1, wherein in step b) the isoelectric precipitation is made at a pH of between 3.0 and 4.0.

4. The procedure according to claim 1, wherein in step c) the pH of the solution is adjusted to pH 6.0.

5. The procedure according to claim 1, wherein in step c) the temperature is 50° C.

6. The procedure according to claim 1, wherein the enzymatic hydrolysis to the extraction step c) comprises the use of alpha-amylase at a temperature higher than room temperature.

7. The procedure according to claim 6, wherein the temperature is 55° C.

8. The procedure according to claim 1, wherein in step d) the homogenous mixing has up to 50% water, up to 10% starch, up to 10% peptide and up to 10% maltodextrins.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Normal Probability Chart for the experimental design used in the development of the current project, which allows to observe the significance of the studied factors.

(2) FIG. 2. Optimization polynomial for protein extraction.

(3) FIG. 3. Optimal values for each of the studied factors in quinoa protein extraction.

(4) FIG. 4. Mass balance for products obtained at the end of the protein extraction process from quinoa flour. (A) Lipid extraction stage not considered, (B) Lipid extraction considered. The percentages correspond to the amount of constituent in each fraction, where the total amount of constituent equals 100%. Calculation base: 100 g commercial quinoa flour.

(5) FIG. 5. Contents of protein, ash, fat and carbohydrates in whole flour (A) compared to lipid-free flour (B), protein and lipid-free flour (C), and protein-free flour(D).

(6) FIG. 6. Relative abundance diagram of peptides released during quinoa protein extract enzymatic digestion (MW: molecular weight, Da)

(7) FIG. 7. Spectro-photometric measurement of reducing sugars released during quinoa starch hydrolysis.

(8) FIG. 8. Peptides and maltodextrins extraction from quinoa flour (flowchart).

(9) FIG. 9. Product formulation.

DETAILED DESCRIPTION OF THE INVENTION

(10) The invention describes the elaboration of foodstuff in gel format produced from quinoa starch, enriched with peptides and maltodextrins obtained from partial protein and starch hydrolysis respectively from the same quinoa grains intended for consumption by sports practitioners during and after physical activity.

(11) The use of protein Chenopodium quinoa for the development of nutrition and food sources has huge growth possibilities. That is why different methods for protein extraction from quinoa have been described. Aluko and Monu (Aluko, R E and Monu, E, Functional and Bioactive Properties of Quinoa Seed Protein Hydrolysates. Journal of Food Science 68:1254-1258 (2003)) detail one of the most widely used methodologies for protein extraction: extraction by alkaline solution, which for the purposes of the present invention was modified and optimized using experimental design, as described next. This extraction method is economic, easy to implement and to be industrialized. As a byproduct from protein extraction, starch is obtained which in turn can be enzymatically hydrolyzed to obtain maltodextrins and monosaccharides useful in the elaboration of new foodstuff.

(12) The first step is to establish the granulometry of quinoa flour. Quinoa commercial flour has a size distribution that goes from 100 μm to 700 μm. We propose to work with granulometry between 100 and 300 μm, which represents 30% total weight of commercial quinoa flour. With bigger granulometry protein extraction becomes inefficient due to the reduction of the extraction surface, while the presence of protein in granulometry smaller than 100 μm is too low. Flour in that size range is composed mainly by starch granules with low protein content.

(13) In order to determine protein concentration in different extraction stages, proximate analysis were made to flour samples during different stages of the process, i.e. quinoa without lipids, quinoa without proteins and quinoa without lipids or proteins. The same analysis was also made to Ecuadorian quinoa grains taking information given by the United States Department of Agriculture (USDA) as reference. Those analyzes were conducted following the methodology proposed by the Association of Official Analytical Chemist (AOAC, Official Methods of Analysis of AOAC International. 16th ed. Washington, D.C. (1995)). Moisture content (drying in an oven at 105° C. for 24 hours), as well as protein (Kjeldahl method*5.7), ash (calcination in muffle at 550° C.) and fat (Soxhlet extraction) was determined in all different samples. Non-nitrogenous extractives (NNE), which correspond to total carbohydrates were determined by difference. The results, expressed in g/100 g of the sample are shown in Table I. The proximate analysis of different quinoa flour samples show that the obtained values in each of the analysis are within previously reported ranges, using proximate composition of a raw Ecuadorian grain and the information described in the USDA row in Table I as pattern. In the case of flour without proteins and flour without lipids or proteins, the increase in moisture content is the result of these components extraction.

(14) The experimental design that was used corresponded to a 2 level factorial design, considering three replicates in the central point. Therefore, the design corresponded to a number 2 (Ruales, J and Nair, B, Nutritional quality of the protein in quinoa (Chenopodium quinoa, Willd) seeds. Plant Foods for Human Nutrition (Formerly Qualitas Plantarum) 42:1-11 (1992)), which generated a total of 19 experiment series in which the optimal conditions for lipid and/or protein extraction were sought. For data analysis, the generation of mathematical models and response optimization, the Design Expert 6.0 software (Stat-Ease Inc, Minneapolis, USA) was used. In the specific case of protein extraction, three factors were evaluated (extraction volume, NaOH concentration and extraction time), which were replicated in flour with 100 μm to 300 μm granulometry, with or without lipids.

(15) To optimize the responses, statistical significance of these effects was evaluated, individually or combined. Results showed that the combined effect between extraction volume and NaOH concentration, as well as the combined effect of NaOH concentration and time, were statistically significant as stated in FIG. 1.

(16) TABLE-US-00001 TABLE I Proximate analysis of quinoa flour samples. Moisture, Proteins Ash, Fat, NNE (total % (N*5,7), % % % carbohydrates), % Samples (bh) (bs) (bs) (bs) (bs) Quinoa 9.7 12.41 2.1 8.19 77.3 flour (100-300 μm) Total Quinoa 9.3 11.57 2.09 1.87 84.47 flour (without lipids) Quinoa 7.6 5.99 2.82 0.7 90.49 flour (without lipids, without proteins) Quinoa 68.3 3.16 2.52 2.52 91.8 flour (with lipids, without) Raw 9.6 16.81 3.65 7.96 71.58 quinoa (Ecuadorian grain)* Quinoa 9.3 14.4 3.2 6.39 76.01 (USDA chart grain)* *Referential flour

(17) A mathematical model representative of the effect of significant factors was generated in order to optimize the response, which in this case is the amount of extracted protein from quinoa flour, using the optimal values that each of the analyzed factors should have. The statistically significant polynomial (p<0,0001) that the optimal result gave is shown in FIG. 2, and was obtained from Design Expert software.

(18) From this model it was possible to determine the optimal values for each evaluated factor, which, for the specific case of protein extraction, were 30 mL of NaOH 40 mM per gram of flour in a two-hour extraction process (FIG. 3). The model turned to be significant at a configuration of a 95% confidence (p value<0.0001), with a “desirability” (adjustment parameter model) which turned to be 0.978 in a 0 to 1 scale (1=maximum adjustment).

(19) Therefore, the extraction process was made using 30 mL of a NaOH 40 mM (pH 12.0) solution for each 1 gram of quinoa flour. This suspension is incubated with constant stirring at room temperature for 2 hours.

(20) Once the extraction is completed, the suspension is centrifuged at 3.000 g for 5 min at 4° C. recovering the supernatant, which contains the soluble quinoa proteins at a concentration between 0.4% and 0.3% w/v, depending if flour with or without lipids was respectively used. This procedure allows obtaining up to an 82% of proteins in the flour. An optional stage is to repeat the protein extraction step on the centrifugation precipitate, which allows recovering an additional ˜8%, thus reaching a 90% protein at the end of the process. Incorporating ionic or non-ionic detergents does not significantly affect extraction efficiency.

(21) Having completed the centrifugation process it is necessary to concentrate the supernatant solution that contains the quinoa proteins, as a previous step to enzymatic hydrolysis. For that purpose there are a number of alternatives: i) carrying out a vacuum concentration so as to avoid protein functionality loss as a result of a denaturation associated to severe thermal treatments, which could affect the peptides properties, ii) applying a nanofiltering technique with pore size membranes <5 kDa, which allows separating and concentrating the proteins in the solution, or iii) isoelectrically precipitating the protein content by adjusting the pH to 3.0 to 4.0 with HCl. However, it must be said that by isoelectric precipitation only ˜60% proteins are recovered, while the other 40% remains in the solution. Proximate analysis performed on the isoelectric precipitate revealed that that it is composed by 75% w/w protein, 2.3% w/w ash, 9.1% w/w lipid (when using flour with lipids) and 14% w/w other constituents (starch, sugar, fiber, etc). Still, the goal of the concentration stage is to reach a protein content of ˜8% w/v in the solution.

(22) Lipids in the quinoa flour used during the development of this research corresponded to an 8.2% of its dry weight (Chart I). An optional prior operation to protein extraction is to remove those lipids so they will not interfere with the final analysis and/or to obtain a more efficient protein extraction. Through experimental design the use of a solution of 95% ethanol in 2:1 volume/weight ratio (mL/g) regarding the amount of quinoa flour (p<0.05) was determined. At smaller volumes of ethanol the suspension becomes very viscous, making it difficult to keep it homogenous during the extraction. Once the ethanol volume has been added, the suspension is hermetically sealed in order to avoid solvent evaporation. Finally, the suspension is incubated under stirring at 30° C. for about 2 hours. Stirring is an important factor to optimize extraction. It must keep flour suspended and avoid decantation during the process. Once the extraction is completed the lipid-free flour is recovered by filtering and by washing it with 95% ethanol. The lipid-free flour is kept at 60° C. all night (>12 hours). This procedure allows extracting around 80% of lipids in the quinoa flour.

(23) Studies highlight the importance of extracting lipids as a stage prior to protein extraction. However, our research has shown that the efficiency of protein extraction increases significantly if this procedure is omitted (FIG. 4). The lipid extraction process drags with it hydrophobic proteins and lipoproteins that may represent up to a 15% of total proteins in quinoa seeds. As a consequence, in the remaining fraction after extraction with NaOH only ˜63% of protein content in lipid-free flour is recovered. This represents ˜20% less protein compared to the efficiency obtained from flour without previous lipid extraction (FIGS. 4 and 5). This result is very important concerning this invention, since it represents an essential difference to other methods of protein extraction described in previous studies. It is also of crucial importance regarding the ultimate goal of protein extraction, which is to obtain protein hydrolysates to be used in the elaboration of foodstuff. The absence of lipid extraction allows us to obtain quinoa peptides at higher levels. These peptides can then be incorporated in a gel for sportspeople in an innovative product formulation with unique functional and nutritional characteristics, and with a clear inventiveness level.

(24) An aminoacid profiling performed on the quinoa protein concentrate (Table II), through high performance liquid chromatography (HPLC) coupled with UV detection allows to clearly observe the nutritional value of quinoa seed, since it is a good source of essential aminoacids such as Arginine (15.3 mg/100 g), Valine (7.4 mg/100 g), Leucine (7.1 mg/100 g), Lysine (6.6 mg/100 g) and sulfur amino acids such as Cysteine (5.5 mg/100 g) and Methionine (5.1 mg/100 g). It should also be considered the presence of other non essential aminoacids whose contribution continues to be significant, like glutamic acid (24.3 mg/100 g), aspartic acid (11.6 mg/100 g) and glycine (10.5 mg/100 g).

(25) TABLE-US-00002 CHART II Aminoacid profile in quinoa protein concentrate. Amino Concentration Amino Concentration acid (mg/100 g) acid (mg/100 g) Asp 11.6 Tyr 3.6 Glu 24.3 Val 7.4 Ser 6.1 Met 5.1 Gly 10.5 Cys 5.5 His 4.0 Ileu 3.3 Arg 15.3 Leu 7.1 Thr 4.4 Phe 4.7 Ala 7.8 Lys 6.6 Pro 9.6

(26) To obtain peptides from proteins extracted from quinoa flour the protein suspension (˜8% w/v) is heated at 80° C. for 5 min and then cooled at 55° C. keeping it for 1 min before adjusting the ph to 8.0 to 8.5 with NaOH or HCl, as appropriate. Then a 0.05 Alcalase Anson Unit (AU) per 1 g of total protein is added to the solution. The solution is incubated at 50 to 60° C. continuously controlling pH. When pH falls below 7.0 then 0.03 AU of a second commercial enzyme (Protamex, Neutrase or Flavourzyme) per 1 g of protein is added and the solution is incubated at 50° C. for 15 to 60 min. To stop the reaction the solution is heated again up to 85° C. for 15 min.

(27) The degree of hydrolysis of the extracted and hydrolyzed protein through the aforementioned process, as well as the molecular weights of the obtained peptides was analyzed using Gel Permeation Chromatography (GPC) (FIG. 6). The results showed that the highest percentage of peptides is within the molecular weight ranging between 1000 and 500 Da.

(28) It is also possible to obtain maltodextrins from quinoa starch through enzymatic treatment. To do that 20 g of quinoa flour with <<100 μm granulometry are prepared. Then 100 mL water is added at room temperature. It is necessary to adjust pH to 6.0 in the solution and then add CaCl.sub.2 at a concentration of 0.05%. Then add 0.4 U/ml of alpha-amylase enzyme. The initial solution is very viscous, but after adding the enzyme it starts to liquefy gradually, evidencing a decrease in the starch molecular weight. The temperature must be increased to 55° C. and then left in incubation for 1 hour under constant stirring. The reaction is stopped by heating the solution to 85° C. for 15 minutes. This stage is very important in order to turn starch into gel later. The amount of reducing sugars released during the process was measured using the Somogyi-Nelson method (Somogyi, M. 1952. Notes on sugar determination. Journal of Biological Chemistry 195: 19-23.). The results are shown in FIG. 7.

(29) It has been observed that the reducing sugar content increases as the reaction time elapses. However, this behavior varies after 15 min reaction, when the hydrolyzing kinetics decreases. Thus, extending starch hydrolysis longer than 20 minutes has no significant effect on the reducing sugar content.

(30) Starch Gel Preparation and Final Product Formulation

(31) The base formulation for quinoa starch gel containing peptides and maltodextrins from the same source is detailed in Table II.

(32) The necessary amount of quinoa flour is weighed (10%) with <<100 μm granulometry, which—as it has been mentioned—is formed mainly by starch. Then the necessary amount of peptides is added to a final 10% concentration and then the maltodextrins to a final 20% concentration. During this stage all the necessary colorants and flavors should be added in order to make the product more attractive. As a preservative, 1 g/kg sorbic acid is added, and then the necessary volume of distilled water. The resulting solution is heated at 80° C. for 20 min under constant stirring in order to turn the starch into gel. Once the desired consistency has been obtained the product is cooled at room temperature. When the mixture has turned into a viscous gel it can be sized in smaller fragments to finally seal it in a suitable packaging.

(33) TABLE-US-00003 TABLE II Base formulation for quinoa starch gel containing peptides and maltodextrins from the same source. Constituent Content (% w/w) Water 50 Peptides 10 Maltodextrins 20 Starch 10 Others (colorants, 10 flavors, etc.)