Acid-hydrolyzed phenolic extract of parsley (Petroselinum crispum L.) leaves inhibits lipid oxidation in soybean oil-in-water emulsions.

The antioxidant activity of the natural phenolic extracts is limited in particular food systems due to the existence of phenolic compounds in glycoside form. Acid hydrolysis post-treatment could be a tool to convert the glycosidic polyphenols in the extracts to aglycones. Therefore, this research investigated the effects of an acid hydrolysis post-treatment on the composition and antioxidant activity of parsley extracts obtained by an ultrasound-assisted extraction method to delay lipid oxidation in a real food system (i.e., soybean oil-in-water emulsion). Acid hydrolysis conditions were varied to maximize total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. When extracts were exposed to 0.6 M HCl for 2 h at 80 ℃, TPC was 716.92 ± 24.43 µmol gallic acid equivalent (GAE)/L, and DPPH radical scavenging activity was 66.89 ± 1.63 %. Not only did acid hydrolysis increase the concentrations of individual polyphenols, but it also resulted in the release of new phenolics such as myricetin and gallic acid. The extract's metal chelating and ferric-reducing activity increased significantly after acid hydrolysis. In soybean oil-in-water emulsion containing a TPC of 400 µmol GAE/L, the acid-hydrolyzed extract had an 11-day lag phase for headspace hexanal compared to the 6-day lag phase of unhydrolyzed extract. The findings indicated that the conversion of glycosidic polyphenols to aglycones in phenolic extracts can help extend the shelf-life of emulsion-based foods.

Insights and Perspectives on Plant-Based Beverages.

The emerging demand for everyday food substitutes is increasing on a daily basis. More and more individuals struggle with allergies and intolerances, which makes it mandatory to provide alternatives for simple products like dairy milk. Plant-based beverages (PBBs) are currently trending due to the multiple diets that promote their consumption with or without a justification. PBBs can derive from various types of plants, not exclusively nuts. Some of the most well-known sources are almonds, soy, rice, and hazelnuts, among others. In view of the need for sustainable approaches to resource utilization and food production, novel sources for PBBs are being sought, and those include fruit kernels. The plant kingdom offers a palette of resources with proven bioactivity, i.e., containing flavonoids, phenolic acids, vitamins, carotenoids, and phenolics, among others. Many of these beneficial substances are water soluble, which means they could be transferred to the plant beverage compositions. The current review aims at comparing the vast number of potential formulations based on their specific nutritional profiles and potential deficiencies, as well as their expected health-promoting properties, based on the raw material(s) used for production. Special attention will be given to the antinutrients, usually abundant in plant-based sources.

Beta-Glucans of Cereals: Functional and Technological Properties.

ß-glucans are a polymeric dietary fiber characterized by ß-(1,3) and ß-(1,4) glycosidic bonds between glucose monomers. They are often used as thickeners, stabilizers, and fat substitutes in foods. The functional and technological quality of ß-glucans is attributed to their origin/source, molecular weight, and structural properties. In particular, physical treatments such as drying, cooking, freezing, and refrigeration influence their molecular, morphological, and rheological characteristics. In addition to their useful technical qualities, ß-glucans are recognized for their numerous beneficial impacts on human health. For this reason, the European Food Safety Authority (EFSA) has provided a positive opinion on health claims such as cholesterol lowering and hypoglycemic properties relating to oats and barley ß-glucans. This paper provides insight into the properties of ß-glucans and different treatments affecting their characteristics and then reviews the latest research on ß-glucans as a functional ingredient for people with type 2 diabetes mellitus (T2DM).

Chlorophylls as Natural Bioactive Compounds Existing in Food By-Products: A Critical Review.

Chlorophylls are a group of naturally occurring pigments that are responsible for the green color in plants. This pigment group could have numerous health benefits due to its high antioxidant activity, including anti-inflammatory, anti-cancer, and anti-obesity properties. Many food by-products contain a high level of chlorophyll content. These by-products are discarded and considered environmental pollutants if not used as a source of bioactive compounds. The recovery of chlorophylls from food by-products is an interesting approach for increasing the sustainability of food production. This paper provides insight into the properties of chlorophylls and the effect of different treatments on their stability, and then reviews the latest research on the extraction of chlorophylls from a sustainable perspective.

Impact of Agronomic Treatments on the Enzymatic Browning of Eggplants (Solanum melongena L.).

Enzymatic browning could negatively affect the sensory and nutritional properties of eggplants post-harvest. Polyphenols, polyphenol oxidase (PPO), and reactive oxygen species (ROS) are three material conditions involved in enzymatic browning. This paper seeks to evaluate the effect of fertilization techniques and grafting on the activity of PPO and colorimetric parameters in cultivated eggplants. Fertilization alone significantly increased the PPO activity in all eggplant fleshes (p ≤ 0.05), whereas the grafting technique combined with fertilization decreased the PPO activity in most of the samples significantly (p ≤ 0.05). Moreover, there was a significant positive correlation between the PPO activity and the a* values of the eggplants. The a* values in grafted eggplants were significantly different from each other (p ≤ 0.05), showing that grafting the fertilized eggplants could be effective in controlling the enzymatic browning. The eggplant slices exposed to air for 60 min at room temperature showed a significant increase (p ≤ 0.05) in PPO activity, browning index (BI), total color difference (ΔE), and a*, b*, and c* values. Thus, it is necessary to minimize the exposure time of the slices to air at room temperature, even if combining fertilization techniques with grafting could delay the enzymatic browning in fresh-cut eggplants.

The α-Amylase and α-Glucosidase Inhibition Capacity of Grape Pomace: A Review.

The concept of functional foods is gaining more importance due to its role in maintaining a healthy status and preventing some metabolic diseases. The control of diabetes, in particular type-2 (T2DM), could be considered a big challenge since it involves other factors such as eating habits. From the pharmacological point of view, inhibiting digestive enzymes, such as α-amylase and α-glucosidase, is one of the mechanisms mainly used by synthetic drugs to control this disease; however, several side effects are described. For that reason, using bioactive compounds may appear as an alternative without presenting the complications synthetic drugs available on the market have. The winemaking industry generates tons of waste annually, and grape pomace (GP) is the most important. GP is recognized for its nutritional value and as a source of bioactive compounds that are helpful for human health. This review highlights the importance of GP as a possible source of α-amylase and α-glucosidase inhibitors. Also, it is emphasized the components involved in this bioactivity and the possible interactions among them. Especially, some phenolic compounds and fiber of GP are the main ones responsible for interfering with the human digestive enzymes. Preliminary studies in vitro confirmed this bioactivity; however, further information is required to allow the specific use of GP as a functional ingredient inside the market of products recommended for people with diabetes.

Impact of Sample Pretreatment and Extraction Methods on the Bioactive Compounds of Sugar Beet (Beta vulgaris L.) Leaves.

To find the most optimal green valorization process of food by-products, sugar beet (Beta vulgaris L.) leaves (SBLs) were freeze-dried and ground with/without liquid nitrogen (LN), as a simple sample pretreatment method, before ultrasound-assisted extraction (UAE) of polyphenols. First, the water activity, proximate composition, amino acid (AA) and fatty acid (FA) profiles, and polyphenol oxidase (PPO) activity of dried and fresh SBLs were evaluated. Then, conventional extraction (CE) and UAE of polyphenols from SBLs using water/EtOH:water 14:6 (v/v) as extracting solvents were performed to determine the individual and combined effects of the sample preparation method and UAE. In all the freeze-dried samples, the specific activity of PPO decreased significantly (p ≤ 0.05). Freeze-drying significantly increased (p ≤ 0.05) the fiber and essential FA contents of SBLs. The FA profile of SBLs revealed that they are rich sources of oleic, linoleic, and α-linolenic acids. Although freeze-drying changed the contents of most AAs insignificantly, lysine increased significantly from 7.06 ± 0.46% to 8.32 ± 0.38%. The aqueous UAE of the freeze-dried samples without LN pretreatment yielded the most optimal total phenolic content (TPC) (69.44 ± 0.15 mg gallic acid equivalent/g dry matter (mg GAE/g DM)) and excellent antioxidant activities. Thus, combining freeze-drying with the aqueous UAE method could be proposed as a sustainable strategy for extracting bioactive compounds from food by-products.

Neither Incretin or Amino Acid Responses, nor Casein Content, Account for the Equal Insulin Response Following Iso-Lactose Loads of Natural Human and Cow Milk in Healthy Young Adults.

Human milk contains <50% less protein (casein) than cow milk, but is equally effective in insulin secretion despite lower postingestion hyperaminoacidemia. Such potency of human milk might be modulated either by incretins (glucagon-like polypeptide-1,GLP-1); glucose-inhibitory-polypeptide, GIP), and/or by milk casein content. Healthy volunteers of both sexes were fed iso-lactose loads of two low-protein milks, i.e., human [Hum] (n = 8) and casein-deprived cow milk (Cow [↓Cas]) (n = 10), as well as loads of two high-protein milks, i.e., cow (n = 7), and casein-added human-milk (Hum [↑Cas]) (n = 7). Plasma glucose, insulin, C-peptide, incretins and amino acid concentrations were measured for 240'. All milks induced the same transient hyperglycemia. The early [20'−30'] insulin and C-peptide responses were comparable among all milk types apart from the low-protein (Cow [↓Cas]) milk, where they were reduced by <50% (p < 0.05 vs. others). When comparing the two high-protein milks, GLP-1 and GIP [5'−20'] responses with the (Hum [↑Cas]) milk were lower (by ≈2−3 fold, p < 0.007 and p < 0.03 respectively) than those with cow milk, whereas incretin secretion was substantially similar. Plasma amino acid increments largely reflected the milk protein content. Thus, neither casein milk content, nor incretin or amino acid concentrations, can account for the specific potency of human milk on insulin secretion, which remains as yet unresolved.

Study of the phenolic profile of a grape pomace powder and its impact on delaying corn oil oxidation.

The grape pomace, the main by-product from the winemaking industry, contains many bioactive substances that must be valorized. The aim of this study was to assess the total phenolic content (TPC), phenolics profile by using HPLC and the antioxidant activity (AOA). The results showed a TPC of 38.86 ± 5.22 g gallic acid equivalent (GAE)/kg while an AOA of 247.84 ± 18.65 µmol Trolox equivalent (TE)/g. Epicatechins were the most representative phenolic compound, according to the HPLC analysis. Then, the grape pomace powder (GPP) was tested in the Rancimat equipment as a natural antioxidant for delaying the corn oil oxidation. Results showed statistically significant differences between the corn oil treated with GP and the control, so the GPP could be a promising natural antioxidant to tackle the oxidation vulnerability of corn oil.

The Perspective of Nectarine Fruit as a Sugar Substituent in Puddings Prepared with Corn and Rice Starch.

It has been long recognized that fruits are healthy diet compounds as they are excellent sources of health-beneficial bioactive components (polyphenols, minerals, vitamins, organic acids, etc.). The diversification of the consumer's taste calls for an expansion of food options and novel ingredients. Puddings are a well-known food choice introduced in the human diet at a very early age because of their easy and high digestion. Four formulations with two types of starch (corn and rice) were selected as object of analysis. Nectarines were incorporated as a purée, and lyophilized powder. The nectarine variety "Gergana", used for the preparations, is a local variety with proven beneficial properties. The study aimed at analyzing the physical (moisture, ash, color, water-holding capacity, water activity, density and syneresis), textural (firmness, gumminess, cohesiveness, springiness, and chewiness), nutritional, and sensory characteristics of the nectarine-enriched puddings. The outcomes obtained from this study provided significant information about the possible application of the formulations in the children's daily menus. All four formulations had distinct peachy aroma. The formulations prepared with nectarine purée resulted in a better sensory perception about their texture, and better water-holding capacity. At this point, the formulation prepared with lyophilized fruit and rice starch has the most promising results. Sufficient evidence leads to further exploration of the perspective of fruit-enriched puddings in order to improve their technological and health-promoting properties.

Effect of Reversal of Whey-Protein to Casein Ratio of Cow Milk, on Insulin, Incretin, and Amino Acid Responses in Humans.

SCOPE: Milk-proteins, besides lactose, stimulate insulin and incretin secretion. Although whey-proteins (WP) are more efficient than casein (Cas) in hormone secretion, the effects of reversal of the (WP/Cas) ratio in whole-milk are poorly known. METHODS AND RESULTS: Healthy volunteers received two different cow-milk drinks, at identical lactose (0.36 g × kg-1 BW) and total-protein (0.18 g × kg1 BW) loads, but at reversed WP/Cas ratio. One is cow-whole milk with a ≈20/80 [WP/Cas] ratio, the other an experimental cow-milk with a ≈70/30 [WP/Cas] ratio ([↑WP↓Cas]-milk). Both milk-types induced the same mild hyperglycemic response. Following [↑WP↓Cas]-milk, the [20'-90'] insulin incremental area (iAUC) (+ ≈44%, p < 0.035), and the [20'-120'] C-peptide iAUC (+ ≈47%, p < 0.015) are greater than those with cow-milk. Similarly, following [↑WP↓Cas]-milk, the GLP-1 [20'-90'] iAUC (+96%, p < 0.025), and the GIP [30'-60'] iAUC (+140%, p < 0.006), were greater than those with cow-milk. Plasma total and branched-chain amino acids are also greater following the [↑WP↓Cas] than cow-milk. CONCLUSIONS: Reversal of the (WP/Cas) ratio in cow-milk enhanced the insulin response, an effect possibly mediated by incretins and/or amino acids(s). These data may be useful in designing specific milk formulas with different effects on insulin and incretin response(s).

GC-MS Metabolic Profile and α-Glucosidase-, α-Amylase-, Lipase-, and Acetylcholinesterase-Inhibitory Activities of Eight Peach Varieties.

The inhibition of certain digestive enzymes by target food matrices represents a new approach in the treatment of socially significant diseases. Proving the ability of fruits to inhibit such enzymes can support the inclusion of specific varieties in the daily diets of patients with diabetes, obesity, Alzheimer's disease, etc., providing them with much more than just valuable micro- and macromolecules. The current study aimed atidentifying and comparing the GC-MS metabolic profiles of eight peach varieties ("Filina", "Ufo 4, "Gergana", "Laskava", "July Lady", "Flat Queen", "Evmolpiya", and "Morsiani 90") grown in Bulgaria (local and introduced) and to evaluate the inhibitory potential of their extracts towards α-glucosidase, α-amylase, lipase, and acetylcholinesterase. In order to confirm samples' differences or similarities, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were also applied to the identified metabolites. The results provide important insights into the metabolomic profiles of the eight peach varieties and represent a first attempt to characterize the peels of the peach varieties with respect to α-glucosidase-, α-amylase-, lipase-, and acetylcholinesterase-inhibitory activities. All of the studied peach extracts displayed inhibitory activity towards α-glucosidase (IC50: 125-757 mg/mL) and acetylcholinesterase (IC50: 60-739 mg/mL), but none of them affected α-amylase activity. Five of the eight varieties showed inhibitory activity towards porcine pancreatic lipase (IC50: 24-167 mg/mL). The obtained results validate the usefulness of peaches and nectarines as valuable sources of natural agents beneficial for human health, although further detailed investigation should be performed in order to thoroughly identify the enzyme inhibitors responsible for each activity.

Impact of In Vitro Gastrointestinal Digestion on the Bioaccessibility of Phytochemical Compounds from Eight Fruit Juices.

Fruits contain a number of useful substances including antioxidants. Their bio-accessibility after passing through the digestive tract is of primary importance when considering their benefits. In this respect, we investigated the effect of in vitro digestion on the phytochemicals of eight fruit juices. Freshly prepared juices from pomegranate, orange and grapefruit were used as well as commercially available juices from cherry, black grapes and aloe vera, blackberry and chokeberry, and two types of chokeberry and raspberries. Spectrophotometric and HPLC methods were used in order to analyse the sugar content, the total phenolic (TPC) and flavonoid contents (TFC), anthocyanins, phenolic acids and antioxidant activity. Principle component analysis was used to explain the differentiation among the types of fruit juice. Sugar recovery variation was between 4-41%. The bio-accessibility of TPC ranged from 13.52-26.49% and of flavonoids between 24.25-67.00%. The pomegranate juice and the juice of black grapes and aloe vera kept 58.12 and 50.36% of their initial anthocyanins content, while for the other samples less than 1.10% was established. As a result, a maximum of 30% remaining antioxidant activity was measured for some of the samples, but for most this was less than 10%. In conclusion, fruit juices are a rich source of biologically active substances, but a more detailed analysis of food transformation during digestion is needed.

Comparative Study of Early- and Mid-Ripening Peach (Prunus persica L.) Varieties: Biological Activity, Macro-, and Micro- Nutrient Profile.

Exploring the chemical composition and biological activity of different fruit varieties is essential for the valorization of their health claims. The current study focuses on a detailed comparative analysis of three early- and two mid-ripening peach varieties: "Filina" (peach), "July Lady" (peach), "Laskava" (peach), "Gergana" (nectarine), and "Ufo 4" (flat peach). They were characterized in terms of essential nutrients such as carbohydrates (sugars and dietary fibers), amino acid content, and lipids as well as mineral content, fat-soluble vitamins, carotenoids, and chlorophyll. Polyphenolic compounds and the related antioxidant activity were also assessed. The methanolic extract of the peel seems to be richer in the studied biologically active substances compared to the fleshy part of the fruit. Anthocyanins were most abundant in "Gergana" and "July Lady" extracts (6624.8 ± 404.9 and 7133.6 ± 388.8 µg cyanidin-3-glucoside/100 g fw, resp.). The total phenol content of the samples varied from 34.11 ± 0.54 to 157.97 ± 0.67 mg gallic acid equivalents (GAE)/100 g fw. "Filina" and "July Lady" varieties possessed the highest antioxidant activity. Overall, the results of this study confirm that the studied peach varieties have satisfactory nutritional value and are potential sources of biologically active substances. Each variety represents an individual palette of nutrients that should be considered separately from the other.

Monitoring the antioxidant activity of an eco-friendly processed grape pomace along the storage.

The aims of this research are to compare and to monitor two conditions for preserving the total phenolic content (TPC) and the antioxidant activity (AOA) of grape pomace (GP) processed as powder and its corresponding extract at room and freezing temperature, respectively. The highest TPC and AOA were obtained in the GP extracted in a ratio 1:10 (w/v) with ethanol at 50% for 45 min at 50 °C. After 9 months of room temperature (RT) storage, the GP powder obtained a significantly higher AOA than the initial condition and the extract frozen-stored the same time.

Biochemical and functional properties of wheat middlings bioprocessed by lactic acid bacteria.

The present study aimed to investigate the bioprocessing of wheat middlings with different lactic acid bacteria (LAB) in order to improve biological activities of this by-product of wheat flour production. The concentration of lactic acid, reducing sugars, and total phenolics, as well as antioxidant, antibrowning, antibacterial and prebiotic activities of fermented samples were analyzed. All LAB strains were capable to growth on wheat middlings, and pH decreased in the medium associated with lactic acid production during cultivation. Samples inoculated with Lactobacillus plantarum DSM20174 presented the maximum growth, lactic acid concentration above 2 mg/ml, and pH values around 3.8. The amount or reducing sugars decreased after 24 hr growth, except for maltose. Bioprocessed wheat middlings exhibited antioxidant, antibrowning, antibacterial, and prebiotic properties, related with the increase of total phenolic content. Highest values for antioxidant activities were obtained for L. plantarum and Streptococcus thermophilus strains, reaching values around 400 and 640 µM Trolox equivalents (TE) ml-1 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) assays, respectively. Bioprocessing techniques using LAB can be an interesting approach to improve the availability of compounds with health-promoting properties from lignocellulosic waste material. PRACTICAL APPLICATIONS: The processing of secondary products from wheat milling can represent an important benefit to the industry. Wheat middlings bioprocessed with LAB showed improved biological activities and may represent an interesting ingredient to be incorporated in food and feed formulations.

Biopreservation of Fresh Strawberries by CarboxymethylCellulose Edible Coatings Enriched with a Bacteriocin from Bacillus methylotrophicus BM47.

Bacteriocins are a large group of antimicrobial compounds that are synthesized by representatives of the genus Bacillus and lactic acid bacteria. They are used extensively in the food industry as biopreservatives. Incorporated in the composition of edible coatings, bacteriocins can reduce microbial growth and decay incidence in perishable fruits, thus improving product shelf-life and commercial appearance. The present study aims to investigate the effect of edible coatings of 0.5% carboxymethyl cellulose (CMC) enriched with a purified bacteriocin from Bacillus methylotrophicus BM47 on the shelf-life extension of fresh strawberries. During storage at 4 °C and 75% relative humidity for 16 days, the measurements of mass loss, decay percentage, total soluble solids (TSS), titratable acidity (TA), pH, organic acids, total phenolic and anthocyanin contents and antioxidant activity were made. The results demonstrate that the application of edible coatings with 0.5% CMC and 0.5% CMC with bacteriocin (CMC+B) led to a significant decrease of mass loss in the treated strawberries compared to the uncoated fruit. After the 8th day of storage, significant reductions in decay percentage along with the absence of fungal growth in CMC+B-coated fruit were observed in comparison with the CMC-coated and control strawberries. During the second half of the storage period, CMC and CMC+B treatments reduced TSS amount in the coated fruit compared to the control, but did not affect the increase of TA and decrease of pH values that are normally associated with postharvest changes. The CMC and CMC+B coatings did not prevent the decrease of ascorbic acid, and total phenolic and anthocyanin contents during cold storage. The application of CMC and CMC+B coatings had a significant inhibitory effect on decreasing the antioxidant activity throughout the storage period and maintained the antioxidant levels in both treatments close to the initial value of 76.8 mmol Trolox equivalents per 100 g of fresh mass.

Valorisation of Ginger and Turmeric Peels as Source of Natural Antioxidants.

The antioxidant activity of ginger and turmeric powders derived from freeze-dried rhizomes and peels as well as commercial spices has been evaluated by two spectrophotometric assays based on electron transfer such as DPPH and FRAP. The phenolic characterization has been also carried out by detecting total polyphenols through Folin-Ciocalteu method and HPLC analysing 6-gingerol and curcumin. Ginger and turmeric peels showed greater antioxidant activity than commercial powders because of their higher phenolic yields. Hence, ginger and turmeric peels can be considered a rich source of phytochemicals which contribute to the antioxidant effects of these agro-food wastes.

A Multifunctional Bread Rich in Beta Glucans and Low in Starch Improves Metabolic Control in Type 2 Diabetes: A Controlled Trial.

DESIGN: Functional foods may be useful for people with diabetes. The soluble fibers beta glucans can modify starch digestion and improve postprandial glucose response. We analyzed the metabolic effects of a specifically designed 'functional' bread, low in starch, rich in fibers (7 g/100 g), with a beta glucan/starch ratio of (7.6:100, g/g), in people with type 2 diabetes mellitus. Methods: Clinical and metabolic data from two groups of age-, sex- and glycated hemoglobin-matched diabetic subjects, taking either the functional bread or regular white bread, over a roughly six-month observation period, were retrieved. RESULTS: Bread intake did not change during the trial. The functional bread reduced glycated hemoglobin by ~0.5% (absolute units) vs. pre-treatment values (p = 0.028), and by ~0.6% vs. the control group (p = 0.027). Post-prandial and mean plasma glucose was decreased in the treatment group too. Body weight, blood pressure and plasma lipids did not change. The acceptance of the functional bread was good in the majority of subjects, except for taste. CONCLUSIONS: A starch-restricted, fiber-rich functional bread, with an increased beta glucan/starch ratio, improved long term metabolic control, and may be indicated in the dietary treatment of type 2 diabetes.

Essential amino acids: master regulators of nutrition and environmental footprint?

The environmental footprint of animal food production is considered several-fold greater than that of crops cultivation. Therefore, the choice between animal and vegetarian diets may have a relevant environmental impact. In such comparisons however, an often neglected issue is the nutritional value of foods. Previous estimates of nutrients' environmental footprint had predominantly been based on either food raw weight or caloric content, not in respect to human requirements. Essential amino acids (EAAs) are key parameters in food quality assessment. We re-evaluated here the environmental footprint (expressed both as land use for production and as Green House Gas Emission (GHGE), of some animal and vegetal foods, titrated to provide EAAs amounts in respect to human requirements. Production of high-quality animal proteins, in amounts sufficient to match the Recommended Daily Allowances of all the EAAs, would require a land use and a GHGE approximately equal, greater o smaller (by only ±1-fold), than that necessary to produce vegetal proteins, except for soybeans, that exhibited the smallest footprint. This new analysis downsizes the common concept of a large advantage, in respect to environmental footprint, of crops vs. animal foods production, when human requirements of EAAs are used for reference.