Amino Acid Composition and Physicochemical Properties of Bitter Lupine (Lupinustermis) Seed Flour

Introduction

Protein-energy malnutrition is a widespread problem throughout the world and has both health and economic consequences. It is the most available deficiency disease especially in third world countries [1].The present feature of population growth shows that protein gap may continue to increase in the future and planned studies are needed to solve the problem. It is difficult and expensive to provide adequate proteins of animal origin. An alternative for improving nutrition status is to supplement the diet with plant proteins.  Recently, a special consideration given for the nutritional evaluation of proteins from plant sources[2]

Legumes (poor man’s meat) are of significance in human nutrition since they are rich sources of protein, calories, certain minerals and vitamins [3].  For food intake, in Afro-Asian states, legumes are the major contributors of protein and Calories for economic and cultural reasons. Food legumes are crops of the family leguminosae(also called fabacae).  They are known as grain legumes, as they are grown for their edible seeds.Developing countries need protein-rich grain legumes for their food habit based on cereal diet and scarcity of fertile land [4], [5],[6].

Legumes are rich in protein, fiber, B-vitamins, iron, folate, calcium, potassium, phosphorus, and zin and low in fat. Legumes are similar to meat in nutrients, but with lower iron levels and no animal fats. The high protein and other nutrients content in legumes make them a goodalternative for meat and dairy products. Vegetarians usually substitute legumes for meat.

Although legumes are rich of nutrients, they are low in calories, but make one feels full. They are of considerable value for diabetic people,as they relatively do not increase blood sugar.  The body uses carbohydrates in legumes slowly, over time, providing steady energy.  Legumes as part of a healthy diet, can help to lower blood sugar, blood pressure, heart rate, other heart diseases and diabetes risks.  The fiber and other nutrients, in legumes,are of use to the digestive system, and may even help to hinder cancer[6].

Lupine is a valuable ancient legume, grown at different regions particularly the Mediterranean area and Indian highlands, with use of seeds as food[7], [8].  Lupine seeds, characterized of high protein content, rich in lysine, an essential amino acid and relatively poor in sulfur containing amino acids

Lupine is within twenty legumes used for human nutrition with cowpea (Vignasinensis L.), kidney bean (Phaseolus vulgaris L.) and pea (Pisumsativum L.) as the most consumed[7], [10].   Although legume seeds contain a moderately high amount of protein, calories, certain minerals and vitamins; their use in food and feed is still limited by their low amount of sulfur-containing amino acids, low protein digestibility and the presence of anti-nutritional components [3],[11.

Materials and methods

Materials

Lupine (Lupinustermis) seed, obtained from local Cairo market, Egypt. The seeds, cleaned to remove husks and foreign matter then stored in polyethylene bags in the refrigerator until used.

Preparation of Lupine seed flours

Bitter lupine seeds whole or de-hulled, crushed using household mill (Braun, Germany).  Fine flour, kept in refrigerator until used.

Hulls and kernel percentages

Dry seed samples, weighed and separated manually into hulls and kernels. The percentage of both kernels and hulls was determined on dry basis.  The three samples measured and the average recorded.

Seed weight

Weight of 100 seeds taken at random, in triplicate. The average reported as 100 seeds weight..

Relative density of the seeds

The increase in volume of 200 ml of distilled water placed in a measuring cylinder after immersing 100 seeds of a known weight [12]. Relative density, calculated as follows:

Oil extraction

Ground whole seeds in n– hexane (BP. 40 – 60 ⁰C) at room temperature for 48 h with several solvent changes, followed by evaporation using rotary evaporator.

Specific gravity of extracted oil

Determined using 10 ml pycnometer at 20 ⁰ C, [13].

Viscosity

Viscosity of oil samples was measured as Cm pois using viscometer ICI Cof Research equipment –London at 50 ⁰C.

Proximate chemical composition

Moisture content, protein, using Kjeldahel method; fat, using Soxhlet apparatus; ash (gravimetrically); crude fibre and carbohydrates (by difference) [14].   Means reported on dry weight basis.

Minerals content

Seeds flour samples were digested by concentrated HNO₃ and HCLO₄ (1:1, V/V) for 2 hours (till solution became colorless). Na, Ca and K were estimated using emission flame photometer (Model Corning 410).  Mg, Mn, Fe, Zn and Cu; determined using atomic absorption spectrophotometer (Prekin – Elmer Instrument Model 2380).

Amino acid analysis

Performed on bitter lupine seed flour using amino acid analyzer [15]

Each sample; hydrolyzed with 6 N HCl at 110 ⁰C for 24 hr. Amino acid composition calculated considering the highest value for each amino acid.  Sulfur-containing amino acids; determined after performing acid oxidation. Chemical score was calculated [16].

Amino acids calculated using the reference pattern [17]. The amino acid showing the lowest percentage (limiting amino acid) representing the chemical score. Essential amino acid index (EAAI) was calculated [18] using the amino acid composition of whole egg protein [19]. Protein efficiency ratio (PER) was estimated according to the following regression equation [20]

PER = – 0.488 +0.454 (Leucine) – 0.105 (Tyrosine).

The net protein value (NPV), calculated as follows:

NPV = (The lowest amino acid score ×% protein) /100

Statistical analysis

Data, based on three replicates, subjected to analysis of variance by complete block design (Gomez & Gomez, 1984). Standard deviation evaluated.  Least significance difference (LSD) mean computed and variations at 5% level; probability (P = 0.05).

Results and discussion

Physical properties of bitter lupine seeds flour

Seed index, hull percentage, kernel percentage, relative density, presented in Table (1). Generally, the values obtained in this study were higher than others [21].

Table 1: Physical properties of bitter lupine seeds

Property	Bitter Lupine seed	LSD
Seed index (g)	20.40 ± 1.10	2.50
Hull %	11.60 ± 0.038	0.80
Kernels %	80.30 ± 0.41	0.98
Relative density (g/cm3)	1.01 ± 0.05	0.10

 

Proximate composition

The moisture content of WBLSF, found 7.3 % and 8.0 % for DBLSF.  Crude protein, fat content, crude fibre, ash and total carbohydrates, (on dry basis) (Table 2).

Crude protein content of WBLSF and DBLSF, found 41.5 and 45.0 %; respectively (Table 2).  This agrees with reported findings for protein in pea, common bean, chickpea and lentil (18.5 to 24%) [22]. Protein content in local and improved cowpea legume, widely consumed in Nigeria (22- 26%) [23]. Protein content of bitter lupine seed is clearly higher (Table 2); this difference can be attributed to genetic and environmental factors [24].

Figure 1  Click here to View figure

 

Fat content

The fat content 2.9 % for WBLSF and 1.2 % for or D, (Table 2); in agreement with (2.2 and 1.1 %), [25].

Crude fiber content

Fiber content, 18.0 % for WBLSF and 12. 1 % for DBLSF disagreeing with other values; 8.08% [27].

Ash content: Ash content, 3.9% for WBLSF and 3.7% for  DBLSF(Table 2). Present results agree with those reported for faba bean legume seeds, 3.6% [28].

Total carbohydrates (calculated by difference)

(33.8 %) for WBLSF and 38.1 % for DBLSF (Table 2).  A range of 24 to 37% starch content; reported for cowpea legume seeds [29].

Table 2: Proximate composition of whole and de-hulled bitter lupine seed flour (on dry basis).

 

Means ± standard deviation of three replicates. Means in the same raw with different letters are significantly different (P < 0.05).  LSD = Least significan for t differences.

Minerals content

Macro and micro mineral composition of WBLSF and DBLSF, presented in (Table 3). Significant (P < 0.05) differences, observed between WBLSF and DBLSF.

Legumes are a good source of minerals with moreCacontent than most cereals [30].  Lupine seed flour, can be used as supplement for cereal flour to improve its Ca content.

Table 3: Macro and micro minerals content of whole and de-hulled bitter lupine seed flour (mg /100 g).

Element	Whole bitter lupine seed flour(WBLSF)	De-hulled  bitter lupine seed flour (DBLSF)	LSD
Macro element:
Potassium	534 ± 2.89	630 ± 5.10	8.12
Sodium	116 ± 2.34	189 ± 2.45	4.20
Calcium	338 ± 3.60	450 ± 3.50	5.80
Micro element:
Zinic	2.30 ± 0.03	2.86 ± 0.05	0.12
Iron	5.30 ± 0.10	6.60 ± 0.13	0.20
Copper	1.04 ± 0.05	1.15 ± 0.06	0.05

Means ± standard deviation of three replicates.LSD = Least significant differences.

Amino acid composition

Amino acid composition of the bitter lupine seed flours (BLF), reported as g/16 g N comparing to the[31].

Glutamic acid was the most abundant amino acid in DBLSF 24.60 g / 16 g nitrogen. The second most abundant amino acid, aspartic acid (9.90 g/16 g N).  The amino acid composition of the four legumes (chickpea, cowpea) indicated little variation in the contents of total essential and non-essential amino acids. The most concentrated essential amino acid in lupine seed was leucine with value (6.90 g/16 g N).  Total amino acid and total essential amino acids in lupine seed were 98.09 and 29.90 g /16 g N; respectively. This is within the range of total essential amino acids without histidine.

Table 4: Amino acids composition of de-hulled bitter lupine seed flour, (g/ 16 g nitrogen).

Amino acid	De-hulled bitter lupine seed flour (DBLSF)	FAO/WHO (1973)
Isoleucine	4.00	4.00
Leucine	6.90	7.00
Lysine	4.50	5.50
Cystine	1.00	–
Methionine	0.34	–
Total sulfur amino acids	1.34	3.50
Tyrosine	4.40	–
Phenylalanine	3.60	–
Total aromatic amino acids	8.00	6.00
Threonine	4.00	4.00
Tryptophan	0.99	1.00
Valine	4.10	5.00
Total essential amino acids	33.83	36.00
Histidine	3.93	–
Arginine	8.50	–
Aspartic acid	9.90	–
Glutamic acid	24.60	–
Serine	4.33	–
Proline	4.80	–
Glycine	4.20	–
Alanine	4.00	–
Total non – essential amino acids	64.26	–

 

Arginine content (8.3 %) agrees with that reported for chickpea seed [2]. Glutamic acid, found to be major non-essential amino acid in studied DBLSF (Table 4).

DBLSF showed a high % of total essential amino acid. The values of total sulphur containing amino acids in DBLSF: 1.34 g/16 g N (Table 4).  The ratio of total acidic and basic amino acids in DBLSF found (34.50 : 16.93 g/16 g N); as reported for cowpea seed protein[32]  suggesting protein in the legumes to be in acidic nature.

Table 5: Classification of amino acids (g/16 g nitrogen) found in de-hulled bitter lupine seed flour (DBLSF).

 

Further amino acid analysis, are shown in (Table 5). Total essential amino acid (TEAA) 29.90 % in DBLSF without histidine; essential aromatic amino acid (EArAA) (8.00 g/16 g N).  On the basis of chemical scores, low leucine :isoleucine ratio in DBLSF is desirable as it lead to amino acid balance in cereals that are already high in leucine and low in tryptophan and isoleucine. DBLSF is deficient in sulfur containing amino acids, as are most legume and vegetable proteins.

The first limiting amino acid was total sulfur amino acids and the second limiting amino acid was valine. The basic amino acid (BAA), (16.93 g/16 g N) found to be less than the total acidic amino acids (AAA), (34.5 g/16 g N) (Table 5) indicating that protein is acidic in nature. The Mitchel essential amino acids index (MEAAI) was 56.40%.

Table 6: Estimation of nutritional quality of de-hulled bitter lupine seed flour(DBLSF); based on amino acids composition.

Material	Protein (%)	First limitingAmino acid	Second limitingAmino acid	Third limitingAmino acid	Chemical score %	Net protein value (NPV)	Protein efficiency ratio (PER)
De-hulled bitter lupinseed flour	45.00	Methionine	Cystine	Tryptophan	12.186	0.153	2.67

 

Conclusion

DBLSF showed high levels of crude protein, oil, crude fibre and ash than  other legumes and wheat flour. Bitter lupine, promising legume seeds, especially for areas in the world where soya (which includes a high amount of both protein and oil) is not available. Bitter lupine seed is particularly relevant to compensate deficiencies in lysine and sulphur-containing amino acids, in cereals and grain legumes, respectively.

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