Phenolic antioxidants in beans and their effects on inhibition of radical-induced DNA damage

Four bean varieties with different hull colors (white, brown, red, and black) were extracted with 80% acetone and evaluated for their antioxidant potential. Red, brown, and black bean extracts were able to effectively retain supercoiled DNA against 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced radical damage at the concentration levels (5, 10, 50, and 100 ppm) tested. Delphinidin, cyanidin, and procyanidins B2, C1, C2, and X were identified in red, brown, and black bean hull extracts, whereas white bean hull extract contained only a small amount of flavonoids. Vanillic, caffeic, p-coumaric, ferulic, and sinapic acids were the main phenolic acids identified in bean hull extracts.     

Phenolic acid content of soils from wheat-no till,wheat-conventional till,and fallow-conventional till soybean cropping systems

Soil core (0–2.5 and/or 0–10 cm) samples were taken from wheat no till, wheat-conventional till, and fallow-conventional till soybean cropping systems from July to October of 1989 and extracted with water in an autoclave. The soil extracts were analyzed for seven common phenolic acids (p-coumaric, vanillic,p-hydroxybenzoic, syringic, caffeic, ferulic, and sinapic; in order of importance) by high-performance liquid chromatography. The highest concentration observed was 4 g/g soil forp-coumaric acid. Folin & Ciocalteu's phenol reagent was used to determine total phenolic acid content. Total phenolic acid content of 0- to 2.5-cm core samples was approximately 34% higher than that of the 0- to 10-cm core samples. Phenolic acid content of 0- to 2.5-cm core samples from wheat-no till systems was significantly higher than those from all other cropping systems. Individual phenolic acids and total phenolic acid content of soils were highly correlated. The last two observations were confirmed by principal component analysis. The concentrations were confirmed by principal component analysis, tions of individual phenolic acids extracted from soil samples were related to soil pH, water content of soil samples, total soil carbon, and total soil nitrogen. Indirect evidence suggested that phenolic acids recovered by the water-autoclave procedure used came primarily from bound forms in the soil samples.The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Melissa officinalis: Composition,Pharmacological Effects and Derived Release Systems—A Review

Melissa officinalis is a medicinal plant rich in biologically active compounds which is used worldwide for its therapeutic effects. Chemical studies on its composition have shown that it contains mainly flavonoids, terpenoids, phenolic acids, tannins, and essential oil. The main active constituents of Melissa officinalis are volatile compounds (geranial, neral, citronellal and geraniol), triterpenes (ursolic acid and oleanolic acid), phenolic acids (rosmarinic acid, caffeic acid and chlorogenic acid), and flavonoids (quercetin, rhamnocitrin, and luteolin). According to the biological studies, the essential oil and extracts of Melissa officinalis have active compounds that determine many pharmacological effects with potential medical uses. A new field of research has led to the development of controlled release systems with active substances from plants. Therefore, the essential oil or extract of Melissa officinalis has become a major target to be incorporated into various controlled release systems which allow a sustained delivery.     

Induction of Phenolic Compounds in Two Cultivars of Cucumber by Treatment of Healthy and Powdery Mildew-infected Plants with Extracts of Reynoutria sachalinensis

Accumulation of phenolic compounds (p-coumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester) was followed in susceptible (Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus) cultivars. The objective was to determine whether these compounds played a role in resistance against powdery mildew following a prophylactic treatment with Milsana (leaf extracts from the giant knot weed Reynoutria sachalinensis, polygonaceae). This treatment significantly reduced the incidence of powdery mildew in both cultivars. Phenolic compounds were extracted from leaves. In the hydrolyzed fraction containing phenolic aglycones, levels of p-coumaric, caffeic, and ferulic acids and of p-coumaric acid methyl ester increased in all treatments (with leaf extracts of R. Sachalinensis, powdery mildew, or both) except the control, one or two days after treatment. In the fraction containing free phenolics, from the tested compounds, only ferulic acid showed an increase in cv. Flamingo (tolerant), and was particularly evident following treatments. On the other hand, the amounts of hydroxycinnamic acids increased rapidly in the two cultivars following Milsana treatment, suggesting their role in disease reduction. All compounds showed antifungal activity when tetsed against common pathogens of cucumber (Botrytis cinerea, Pythium ultimum, and P. aphanidermatum), but in general methyl esters were more fungitoxic than their corresponding free acids. This study suggests that cucumber is able to release antifungal compounds that are instrumental in repressing powdery mildew infection. This response is seemingly independent from the level of genetic resistance associated with each cultivar.     

Allelopathic potential in bilberry-spruce forests: Influence of phenolic compounds on spruce seedlings

Regeneration failure ofPicea abies in a subalpine bilberry-spruce forest was studied in relation to phenolic compounds, their occurrence and toxicity. Germination bioassays with natural leachates of bilberry (Vaccinium myrtillus) and spruce showed negative effects on root elongation of spruce seedlings. Growth bioassays on litter and humus demonstrated inhibitory effects of these organic layers.p-Hydroxyacetophenone, a spruce-specific metabolite, was isolated in spruce throughfall (10^–6 M), in water extracts of litter (between 1 and 8 µg/g dry wt) and organic layer (less than 1 µg/g dry wt) in addition to tannins and several common phenolic acids. Potential relationships between vegetation cover and phenolic pattern of the soil are discussed, since organic layers under bilberry heath exhibited higher amounts of phenolic acids and tannins than those under spruce.p-Hydroxyacetophenone and caffeic acid reduced, even at 5 × 10^–5 M, spruce seedling growth, especially root development, with additive effects for these two monomers. Autotoxicity involving spruce trees and allelopathy of understory species, mediated byp-hydroxy-acetophenone and other phenolic compounds, including tannins, deserves further attention in regeneration studies.     

Effect of maturity on phenolics (phenolic acids and flavonoids) profile of strawberry cultivars and mulberry species from pakistan

In this study, we investigated how the extent of ripeness affects the yield of extract, total phenolics, total flavonoids, individual flavonols and phenolic acids in strawberry and mulberry cultivars from Pakistan. In strawberry, the yield of extract (%), total phenolics (TPC) and total flavonoids (TFC) ranged from 8.5-53.3%, 491-1884 mg gallic acid equivalents (GAE)/100 g DW and 83-327 mg catechin equivalents (CE)/100 g DW, respectively. For the different species of mulberry the yield of extract (%), total phenolics and total flavonoids of 6.9-54.0%, 201-2287 mg GAE/100 g DW and 110-1021 mg CE/100 g DW, respectively, varied significantly as fruit maturity progressed. The amounts of individual flavonols and phenolic acid in selected berry fruits were analyzed by RP-HPLC. Among the flavonols, the content of myricetin was found to be high in Morus alba (88 mg/100 g DW), the amount of quercetin as high in Morus laevigata (145 mg/100 g DW) while kaempferol was highest in the Korona strawberry (98 mg/100 g DW) at fully ripened stage. Of the six phenolic acids detected, p-hydroxybenzoic and p-coumaric acid were the major compounds in the strawberry. M. laevigata and M. nigra contained p-coumaric acid and vanillic acid while M. macroura and M. alba contained p-hydroxy-benzoic acid and chlorogenic acid as the major phenolic acids. Overall, a trend to an increase in the percentage of extraction yield, TPC, TFC, flavonols and phenolic acids was observed as maturity progressed from un-ripened to fully-ripened stages.     

Antioxidant activity of almonds and their by-products in food model systems

Antioxidant activities of almond whole seed, brown skin, and green shell cover extracts, at 100 and 200 ppm quercetin equivalents, were evaluated using a cooked comminuted pork model, a β-carotene-linoleate model, and a bulk stripped corn oil system. Retention of β-carotene in a β-carotene-linoleate model system by almond whole seed, brown skin, and green shell cover extracts was 84–96, 74–83, and 71–93%, respectively. In a bulk stripped corn oil system, green shell cover extract performed better than brown skin and whole seed extracts in inhibiting the formation of both primary and secondary oxidation products. In a cooked comminuted pork model system, green shell cover and brown skin extracts inhibited the formation of TBARS, total volatiles, and hexanal more effectively than did the whole seed extract. HPLC analysis revealed the presence of caffeic, ferulic, p-coumaric, and sinapic acids as the major phenolic acids in all three almond extracts examined.     

Analysis of Phenolic Compounds and Antioxidant Abilities of Extracts from Germinating Vitis californica Seeds Submitted to Cold Stress Conditions and Recovery after the Stress

The material for this study consisted of stratified seeds of Vitis californica submitted to germination under optimum conditions (+25 °C) or under chill stress (+10 °C), also followed by recovery. It has been determined that the germinating seeds contain considerable amounts of tannins, catechins as well as phenolic acids such as gallic, p-coumaric, caffeic and ferulic acids. Gallic acid appeared in the highest amount in the germinating seeds (from 42.40–204.00 µg/g of fresh weight (FW)), followed by caffeic acid (from 6.62–20.13 µg/g FW), p-coumaric acid (from 2.59–5.41 µg/g FW), and ferulic acid (from 0.56–0.92 µg/g FW). The phenolic acids occurred mostly in the ester form. Under chill stress, the germinating seeds were determined to contain an elevated total amount of phenolics, as well as raised levels of condensed tannins, catechins, gallic acid, and gafeic acid. The levels of p-coumoric and ferulic acids were found to have decreased. In extracts isolated from a sample exposed to low temperature, increased antioxidant activity and reduction potential were also demonstrated. Tissue of the germinating seeds which underwent post-stress recovery was found to have less total phenolics.     

Evaluation of the Phenolic Content and Antioxidant Activity of Different Seed and Nut Cakes from the Edible Oil Industry

The objective of this work was to extract water‐soluble compounds from different seed and nut cakes under the same conditions and compare the phenolic content and antioxidant activity of the cake extracts. Seed cakes of sunflower, pumpkin, flaxseed and defatted sesame, and nut cakes of almond, pecan, macadamia and hazelnut were used in the experiments. Extracts were obtained by solid–liquid extraction with a water/ethanol solution (20:80, v/v). Total phenolic content, flavonoids, flavan‐3‐ols and condensed tannins in the extracts were determined using spectrophotometric analysis. Antioxidant properties of the extracts were determined by the DPPH and ABTS radical scavenging methods, and by determination of the reducing power and chelating activity. The extract from pecan nut cake presented the highest amounts of all compounds analyzed, followed by sunflower seed and hazelnut cake extracts. These samples also had the highest effects on the ABTS and DPPH radicals, as well as the uppermost reducing powers. The extracts from pecan nut and sunflower and sesame seeds were analyzed using HPLC and individual phenolics were further characterized.     

Antioxidative activity and phenolic composition of pilot-plant and commercial extracts of sage and rosemary

Eight sage (Salvia officinalis) and twenty-four rosemary (Rosmarinus officinalis) extracts, originating from pilot-plant or commercial sources, had different antioxidative activities as measured by accelerated autoxidation of methyl linoleate. Twenty-seven compounds were characterized in the Labiatae family extracts by high-performance liquid chromatography (HPLC) coupled with mass spectrometry, equipped with an atmospheric pressure chemical ionization interface, and by HPLC coupled with a photodiode array spectrophotometer. Twenty-two compounds were identified, including phenolic acids, carnosol derivatives, and flavonoids. The extracts showed great variation in their HPLC profiles, and no correlation was apparent between their antioxidative efficiency and their composition, in twenty specific phenols. Data indicated that the most effective compounds were carnosol, rosmarinic acid, and carnosic acid, followed by caffeic acid, rosmanol, rosmadial, genkwanin, and cirsimaritin.     

Altitudinal Variation of Phenolic Contents in Flowering Heads of <Emphasis Type="Italic">Arnica montana</Emphasis> cv. ARBO: a 3-Year Comparison

In continuation of our studies of altitudinal effects on secondary metabolite profile of flowering heads from taxa of the Asteraceae, we investigated phenolic contents and radical scavenging potential from cultivated plants of Arnica montana cv. ARBO during the growing seasons 2003, 2004, and 2005. By conducting experiments on potted plants, we excluded that differences in phenolic contents from plants grown at different altitudes were related primarily to differences in soil composition at these sites. To assess altitudinal and interseasonal variation, plants of A. montana cultivar ARBO were grown in nine experimental plots at altitudes between 590 and 2,230 m at Mount Patscherkofel near Innsbruck, Austria. In all growing seasons and regardless of the soil the plants were grown in, the proportion of flavonoids with vicinal-free hydroxy groups in ring B to flavonoids lacking this feature, and the total amount of caffeic acid derivatives, significantly increased with elevation. These increases of antioxidant phenolics corresponded to an increase of the radical scavenging potential of extracts from plants grown at different altitudes. The results are discussed in regard to previous studies that suggest that enhanced UV-B radiation and decreased temperatures trigger augmented biosynthesis of UV-absorbing and antioxidant phenolics in higher plants.     

Unexploited Thapsia garganica,Orlaya maritima,and Retama raetam Seeds: Potential Sources of Unsaturated Fatty Acid and Natural Antioxidants

Total lipid contents, fatty acid compositions, phenolic profiles and antioxidants activities of seeds from Thapsia garganica, Orlaya maritima, and Retama raetam were investigated. The oil values were more than 26 %, except seeds of R. raetam (ca. 3 %). Unsaturated fatty acids accounted for the majority of the fatty acids (more than 75 %). Oleic and linoleic acid were the predominant fatty acids. Total phenolic compounds (24–104 mg GAE g^?1 DR), total flavonoids (4–102 mg QE g^?1g DR), total tannins (28–85 mg GAE g^?1 DR) and condensed tannins (0.62–131 mg CE g^?1 DR) were also determined. The antioxidant activities using different assays were evaluated. The predominant detected classes were the phenolic acids (42–85 %) and the flavonoids (11–48 %). The major phenolic acids were caffeic, trans‐4‐hydroxy‐3‐methoxycinnamic, p‐coumaric, and gallic acid. The predominant flavonoids were quercetin, luteolin, naringin, apigenin, and kaempferol. This study brings attention to the medicinal importance of these species as a source of oil and antioxidant molecules.     

Identification and function of antioxidants from oat groats and hulls

Antioxidant components of methanolic extracts of groats and hulls from Ogle oats were identified and quantified by using gas chromatography-mass spectrometry (GC-MS) and gas chromatography after N,O-bis(trimethylsilyl)acetamide derivatization. Ferulic, p-coumaric, vanillic, p-hydroxybenzoic, 4-hydroxyphenylacetic acids and vanillin and catechol were quantified in groat and hull extracts. Additionally, caffeic acid in groat extracts, and o-coumaric, sinapic and slaicylic acids in hull extracts were quantified. Extracts from groats and hulls at levels of 0.05, 0.1, 0.2, and 0.3% w/w, based on total phenolic content, were added to soybean oil, and their antioxidant effectiveness was compared with that of 0.02% w/w tertiary butylhydroquinone (TBHQ) and a control (no additives) at 60°C in the dark by measuring peroxide values. Antioxidant activities of both extract sources increased with increased concentration. During 20 d of storage, the groat extract (0.3%) was not significantly different from TBHQ after day 16, and hull extracts (0.2 and 0.3%) were not significantly different from TBHQ on day 20. Oils containing pure phenolics at the same concentrations measured in the groat and hull extracts oxidized more quickly than did oils containing the extracts.     

Antioxidant effect of the constituents of susabinori (Porphyra yezoensis)

The antioxidant activities of several extracts from Susabinori (Porphyra yezoensis) were measured by the ferric thiocyanate method and the thiobarbituric acid method. The methanol, acetone, ethyl acetate, and hexane extracts, and the chloroform-soluble and water-soluble fractions from the chloroform-methanol extract exhibited higher activities than α-tocopherol. The hot water extract showed little activity. Thin-layer chromatography analysis of the active extracts suggested the existence of several antioxidants. The activity of the chloroform soluble fraction was due to chlorophyll analogs. A strong antioxidant was isolated from the methanol extract, accompanied by several amino acids such as leucine and phenylalanine. This compound was identified as usujilene, a kind of mycosporine-glycine like amino acid.     

Primary Metabolites and Polyphenols in Rapeseed (Brassica napus L.) Cultivars in China

Defatted meals of 10 rapeseed (Brassica napus L.) varieties were investigated for their total phenolic, phenolic acid (free, esterified, and insoluble-bound forms), and tannin contents. The antioxidant capacities (AC) of methanol extracts from samples were assessed using the 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), Folin–Ciocalteu method and ferric reducing antioxidant power (FRAP), and β-carotene–linoleic acid tests. In the fraction of free phenolic acids, sinapic, caffeic, ferulic, syringic, gallic, and p-coumaric acids were identified. In the fraction of esterified phenolic acids, sinapine, sinapoyl glucoside, and disinapoyl gentiobiose were identified. After basic hydrolysis, sinapic, ferulic, cinnamic, and 4-hydroxybenzoic acids were identified, and sinapic acid (SA) constituted 98.3% to 99.6% of the total esterified phenolic acids. Eleven components (sinapic, protocatechuic, p-coumaric, syringic, vanillic, gallic, caffeic, ferulic, salicylic, cinnamic, and 4-hydroxybenzoic acids) in the fraction of insoluble-bound phenolic acids were identified. The AC of the samples correlated with the total phenolic content. Overall, the total phenolics showed a better correlation with AC than the individual phenolic compounds. Moreover, SA, sinapoyl glucoside, and disinapoyl gentiobiose showed a highly significant and strong positive correlation with the AC of rapeseed meals, and the derivatives of cinnamic acid showed a higher correlation with AC than the derivatives of benzoic acid. The change in the canolol content in rapeseeds under microwave irradiation is discussed. The correlation of the canolol formed with SA and its derivatives is discussed.     

Effects of phenolic acids on germination and early growth of herbaceous woodland plants

Four herbaceous plant species from woodland (clearings),Deschampsia flexuosa, Scrophularia nodosa, Senecio sylvaticus, andChamaenerion angustifolium, were tested for their sensitivity to phenolic acids. Seven commonly occurring phenolic compounds were used in a germination experiment in concentrations ranging from 0.01 to 10 mM, i.e., salicylic,p-hydroxybenzoic, syringic, caffeic, vanillic,p-coumaric, and ferulic acids. Germination was delayed rather than inhibited. Radicle elongation was strongly affected; at lower concentrations stimulatory effects were observed, whereas at high concentrations radicle elongation was severely reduced. Salicylic acid was the most effective phenolic compound, whereas caffeic acid caused no effects. Early growth was studied in more detail in a second experiment withDeschampsia flexuosa andSenecio sylvaticus and the phenolic acids, ferulic and p-coumaric acid. Primary root length, number and length of secondary roots, and dry weight were stimulated at 0.01 mM but were inhibited at 10 mM of both compounds. The results are discussed in view of the allelopathic relations between trees and herbaceous understory vegetation.     

Allelopathic activity in wheat-conventional and wheat-no-till soils: Development of soil extract bioassays

The primary objective of this research was to determine if soil extracts could be used directly in bioassays for the detection of allelopathic activity. Here we describe: (1) a way to estimate levels of allelopathic compounds in soil; (2) how pH, solute potential, and/or ion content of extracts may modify the action of allelopathic compounds on germination and radicle and hypocotyl length of crimson clover (Trifolium incarnatum L.) and ivyleaved morning glory (Ipomoea hederacea L. Jacquin.); and (3) how biological activity of soil extracts may be determined. A water-autoclave extraction procedure was chosen over the immediate-water and 5-hr EDTA extraction procedures, because the autoclave procedure was effective in extracting solution and reversibly bound ferulic acid as well as phenolic acids from wheat debris. The resulting soil extracts were used directly in germination bioassays. A mixture of phenolic acids similar to that obtained from wheat-no-till soils did not affect germination of clover or morning glory and radicle and hypocotyl length of morning glory. The mixture did, however, reduce radicle and hypocotyl length of clover. Individual phenolic acids also did not inhibit germination, but did reduce radicle and hypocotyl length of both species. 6-MBOA (6-methoxy-2,3-benzoxazolinone), a conversion product of 2-o-glucosyl-7-methoxy-1,4-benzoxazin-3-one, a hydroxamic acid in living wheat plants, inhibited germination and radicle and hypocotyl length of clover and morning glory. 6-MBOA, however, was not detected in wheat debris, stubble, or soil extracts. Total phenolic acids (FC) in extracts were determined with Folin and Ciocalteu's phenol reagent. Levels of FC in wheat-conventionaltill soil extracts were not related to germination or radicle and hypocotyl length of either species. Levels of FC in wheat-no-till soil extracts were also not related to germination of clover or morning glory, but were inversely related to radicle and hypocotyl length of clover and morning glory. FC values, solute potential, and acidity of wheat-no-till soil extracts appeared to be independent (additive) in action on clover radicle and hypocotyl length. Radicle and hypocotyl length of clover was inversely related to increasing FC and solute potential and directly related to decreasing acidity. Biological activity of extracts was determined best from slopes of radicle and hypocotyl length obtained from bioassays of extract dilutions. Thus, data derived from the water-autoclave extraction procedure, FC analysis, and slope analysis for extract activity in conjunction with data on extract pH and solute potential can be used to estimate allelopathic activity of wheat-no-till soilsThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, or criticism of similar ones not mentioned.     

Antioxidant activity of flavonoids isolated from Scutellaria rehderiana

The present study examined the antioxidant activity in heated canola oil of hexane, acetone, and methanol extracts of dry roots of gansu huangqin (Scutellaria rehderiana) as well as six flavonoids isolated from the acetone and methanol extracts. The oxidation was conducted at 95°C by monitoring oxygen consumption and decreases in both linoleic and α-linolenic acids. The acetone extract was most effective in inhibiting oxidation of canola oil, followed by the methanol extract. The antioxidant activity of gansu huangqin acetone extract was dose-dependent. Among the six flavonoids, baicalein and ganhuangenin were more effective than butylated hydroxytoluene (BHT) in protecting canola oil from oxidation. The present results suggest that the acetone extract of this root may be a potential source of natural antioxidants for use in processed foods.     

<Emphasis Type="Italic">Lesquerella fendleri</Emphasis> protein fractionation and characterization

Lesquerella fendleri is a promising new crop whose seed contains hydroxy FATG with potential industrial uses as well as substantial amounts of valuable gums. The defatted L. fendleri seeds also contain more than 30% protein. The objective of this study is to process and characterize this protein component for possible future uses in food. Hexane-defatted seed has more than 30% protein content. Defatted lesquerella meal was extracted sequentially with 0.5 M sodium chloride (2×), water, 70% ethanol, and 0.1 N sodium hydroxide (2×). Each sodium chloride extract was dialyzed against deionized water and centrifuged to separate the water-soluble fraction (albumin) from the salt-soluble fraction (globulin) before freeze-drying. The ethanol extract and the neutralized sodium hydroxide extracts (glutelin) were dialyzed against water and freeze-dried. Albumin had the highest proportion of lysine and sulfur amino acids per 16 g nitrogen among all the fractions analyzed. Glutelin and globulin accounted for the highest amount of protein nitrogen. SDS-PAGE of the reduced albumin, globulin, and glutelin showed the presence of several protein bands with M.W. ranging from 7 to 98 kDa. Nitrogen solubility of defatted lesquerella meal from pH 2 to 12 indicated a solubility minimum of 15% around pH 4.2 and a solubility of 75% at pH 11.5. Nonprotein nitrogen of defatted meal was 12% of total nitrogen. Defatted lesquerella meal has the potential for food use based on good nitrogen solubility and good amino acid composition.     

Protein fractionation and properties of salicornia meal

Salicornia bigelovii Torr. is an annual salt-marsh oilseed plant. Hexane-defatted salicornia meal was extracted sequentially with 0.5 M sodium chloride (2x), water, 70% ethanol, and 0.1 N sodium hydroxide (2x). Each sodium chloride extract was dialyzed against deionized water and centrifuged to separate a water-soluble fraction (albumin) from a salt-soluble fraction (globulin) before freeze-drying. Ethanol extracts and neutralized sodium hydroxide extracts (glutelin) were dialyzed against water and freeze-dried. Globulin accounted for the highest amount of protein nitrogen, followed by glutelin and albumin. SDS-PAGE of reduced albumin, globulin, and glutelin showed a number of protein bands. Nitrogen solubility of defatted salicornia meal from pH 2 to 11 indicated a minimum solubility of 22%, around pH 4.5. Nonprotein nitrogen of defatted meal was 23% of total nitrogen, higher than defatted soybean, sunflower, and rapeseed meals. Albumin had the highest proportion of lysine and sulfur amino acids per 16 g nitrogen among all the fractions analyzed.