185

SUMMARY Title: Studies on isolation and related biochemical parameters of leaf

protein concentrate from Populus deltoides, Pongamia pinnata and

Albizia lebbeck. The thesis has been organized into five chapters as summarized below:

Chapter I: Introduction This chapter gives an overview of the status of malnutrition caused by nutritional

deficiency, particularly protein deficiency in human beings and animals throughout

the globe in general and in developing world including India, in particular;

consequences of protein malnutrition, reasons for drawing special attention to protein

deficiency; requirement for strengthening the measures, including search of additional

and new protein sources to combat protein deficiency; importance of leaf protein and

its limitation to the monogastric animals; need for developing leaf protein concentrate

(LPC) from different plant sources; and potentialities of tree leaves for production of

protein concentrate. Also presented in this chapter is a detailed account of the LPCs

including their production methods, nutritional characteristics, advantages and use as

a food and feed supplement. Potential of the large quantity of the leaves generated as

plantation by products for their utilization in production of LPCs has also been

realized here.

Chapter II: Review of Literature

This chapter presents an account of the detailed literature survey, on the research and

development of LPCs published globally, conducted by SciFinder, and regular

screening of various published literature. Need of the present work based on the

inferences drawn from review of literature was brought out and objectives were made.

The study was intended to explore the utilization of leaves from some of the forest

plants such as Populus deltoides, Pongamia pinnata and Albizia lebbeck for

production of LPC. The objective of the study was to isolate LPC from these plant

species and to carry out its biochemical assessment. Rationale for selection of tree

species – Populus deltoides, Pongamia pinnata and Albizia lebbeck for the study has

also been given. Chapter III: About the Plant Species This chapter describes the background information on the tree species namely

186

Populus deltoides, Pongamia pinnata and Albizia lebbeck selected for the

investigation. This includes scientific classification, common names, habit and

habitat, distribution, morphological description, biochemical composition of the

leaves, and their uses.

Chapter IV: Materials and Methods This chapter embodies experimental part including (i) plant materials, (ii)

optimization of process conditions for isolation of leaf protein concentrates from

Populus deltoides, Pongamia pinnata and Albizia lebbeck, (iii) isolation of leaf

protein concentrates from lower, middle and upper canopy of Populus deltoides,

Pongamia pinnata and Albizia lebbeck using optimized protocol (iv) Biochemical

assessment of the isolated LPCs from lower, middle and upper canopy of Populus

deltoides, Pongamia pinnata and Albizia lebbeck including ‘Chemicals and

Reagents’, ‘Nutritional composition’, ‘in vitro protein digestibility’, and ‘anti

nutritional factors’, and (v) statistical analysis

Fresh leaves of Populus deltoides (PD), Pongamia pinnata (PP) and Albizia

lebbeck (AL) were collected in the early morning from the trees grown in the campus

of Forest Research Institute, Dehradun, India which lies between latitudes 29°58'N

and 31°2'N and longitudes 77°34'E and 78°18'E. The plant material was authenticated

at Systematic Botany Discipline, Botany Division, Forest Research Institute,

Dehradun, India. The leaves were analyzed for moisture content using oven dry

procedure, and the average dry matter content of the leaves was PD, 34.99±0.42 %;

PP, 39.31±0.32% and AL, 42.11±0.29%. The chemicals and reagents employed in

isolation of the LPCs and their biochemical assessment were of laboratory / analytical

grade, wherever applicable, and procured from the standard suppliers. The

instruments used during the investigation include Electronic balance (Denver

Instruments, India), Hot air oven (Microsil, India), Micro Kjeldahl’s apparatus

(Kjeltec system- 1026), Magnetic stirrer (Remi Motors Ltd, India), Soxhlet apparatus

(Secor India), Flash rotatory evaporator (Medica Instrument Mfg. Co, India), Muffle

furnace (Widsons Scientific Works, India), Vacuum pump (Decibel Instruments,

India), Water Bath (Locally manufactured), pH meter (Eutech Instruments, India),

UV-Spectrophotometer (Thermo Fisher scientific, India), Centrifuge (Kendro

Laboratory Products, Germany), Vortexes (Remi, India), Flame photometer

187

(Systronics India), Mechanical shaker (Remi Motors Ltd, India) and Atomic

absorption spectrophotometer (GBC 904, Electronic Corporation of India).

The LPCs (PDLPC, PPLPC, ALLPC) from the fresh leaves of PD, PP and AL,

respectively were isolated following a procedure comprised of mincing of the fresh

leaves with water, pressing to yield a green juice, coagulating the protein followed by

centrifugation of the coagulum, its washing and drying. However, the procedure for

isolation of the LPCs was optimized by varying the process conditions with respect to

the yield of LPC (expressed in g/100g leaves) and protein content (%). Each of these

parameters was varied one by one keeping the remaining parameters constant. Briefly,

fresh leaves (100g) were washed with tap water, drained and minced with distilled

water (1:1 to 1:11, w/v) in a locally purchased mixer followed by filtration through

muslin cloth. The leaf protein from the separated juice was coagulated by 1N acetic

acid at ambient temperature or by heating after adjusting the pH (acidic pH-4.0, basic

pH-9.0 and bio pH-7.0) for the desired temperature (50°C to 100°C) for fixed duration

(1-15 minutes). The coagulum was centrifuged at 10000 rpm for 10 minutes, washed

with distilled water till neutral pH and oven dried until constant weight. The LPCs

were stored at -200C until analyzed. Further, the LPCs (PDLPCL, PDLPCM,

PDLPCU; PPLPCL, PPLPCM, PPLPCU and ALLPCL, ALLPCM, ALLPCU) were

also isolated from lower, middle and upper canopy of PD, PP and AL, respectively

using the optimized protocol.

PDLPCL, PDLPCM, PDLPCU; PPLPCL, PPLPCM, PPLPCU and ALLPCL,

ALLPCM, ALLPCU were subjected to their biochemical assessment wherein their

nnutritional composition, in vitro protein digestibility, and anti nutritional factors

were determined. Proximate nutritional composition of these LPCs was ascertained by

analyzing the content of moisture, nitrogen, crude protein (N X 6.25), total organic

carbon, C/N ratio, fat, ash, crude fiber, nitrogen free extract, organic matter, total

carbohydrate, total dietary fiber, pigments (chlorophylls, total carotenes and

xanthophylls), minerals (Ca, P, Na, K, Fe, Cu, Zn, Mn, Mg and Co), total free amino

acids and gross energy in these LPCs employing the standard AOAC methods and or

published in literature. The in vitro protein digestibility was determined using the

Pepsin-Trypsin and Pepsin-Pancreatin methods, while anti-nutritional factors such as

total phenolic content, total saponins and total alkaloids were estimated using the

reported procedures.

188

All the experiments were done in triplicate and the mean values so determined

were subjected to one-way analysis of variance (ANOVA) using the SPSS version

16.00. Statistical significance of the differences was performed by post-hoc analysis

using Tukey test. Differences with p < 0.05 were considered significant. For better

interpretation of significant results, critical difference (CD) or least significant

difference (LSD) was determined using Genstsat version 3.2 statistical software.

Chapter V: Results and Discussion The procedure for isolation of PDLPC, PPLPC, ALLPC was optimized with respect

to the yield of LPC (expressed in g) and protein content (%) by varying the process

parameters. Each of these parameters was varied one by one keeping the remaining

parameters constant. The yield of LPC and the protein content of the products

obtained under varied process conditions were determined and their dependence on

each of the variables was investigated. The process parameters thus standardized for

isolation of these LPCs consisted of the ratio of fresh leaves to water (1:9),

coagulation temperature (90ºC) and duration (11 minutes), and pH (4.0) using 1N

citric acid.

Application of the optimized process enabled to isolate the PDLPCL,

PDLPCM, PDLPCU; PPLPCL, PPLPCM, PPLPCU and ALLPCL, ALLPCM,

ALLPCU containing protein 59.24±0.50, 59.56±0.40 and 59.66±0.10%; 43.77±0.23,

43.95±0.34 and 44.34±0.53%; 37.15±0.26, 37.57±0.32 and 37.76±0.68% in

7.79±0.01, 7.66±0.12 and 7.83±0.14%; 7.27±0.08, 7.28±0.04 and 7.34±0.04%;

5.99±0.12, 5.97±0.08 and 6.07±0.12% yield, respectively.

The proximate nutritional composition of PDLPCs (PDLPCL, PDLPCM,

PDLPCU), PPLPCs (PPLPCL, PPLPCM, PPLPCU), and ALLPCs (ALLPCL,

ALLPCM, ALLPCU) was found to be moisture (%), 6.91±0.19, 7.08±0.20,

7.08±0.08, 6.58±0.36, 6.49±0.10, 6.51±0.09, 6.54±0.31, 6.81±0.02, 6.87±0.25; total

organic carbon (%), 34.30±0.56, 34.22±0.79, 35.19±0.86, 40.90±0.34, 40.33±0.92,

41.04±0.64, 52.34±0.16, 52.38±0.23, 52.66±0.47; nitrogen (%), 9.47±0.08,

9.53±0.06, 9.54±0.03, 7.00±0.04, 7.03±0.05, 7.09±0.08, 5.94±0.04, 6.01±0.05,

6.09±0.08; C/N ratio, 3.61±0.08, 3.59±0.09, 3.68±0.09, 5.84±0.05, 5.73±0.15,

5.79±0.08, 8.80±0.08, 8.71±0.11, 8.64±0.19; fat (%),17.96±1.02, 16.95±0.37,

18.27±0.09, 12.54±0.31, 12.72±0.50, 12.82±0.49, 17.29±0.16, 16.97±0.17,

17.29±0.27; crude fiber (%), 1.54±0.07, 1.53±0.05, 1.54±0.03, 1.84±0.05, 1.86±0.06,

189

1.85±0.10, 1.99±0.01, 1.98±0.08, 2.03±0.06; ash (%), 4.35±0.03, 4.38±0.02,

4.36±0.02, 3.85±0.08, 3.97±0.69, 3.66±0.19, 5.48±0.24, 5.30±0.17, 5.37±0.36;

nitrogen free extract (%), 16.89±0.79, 18.03±0.80, 16.76±0.28, 37.99±0.43,

37.49±0.60, 37.32±0.89, 38.08±0.64, 38.17±0.28, 37.56±0.74; total carbohydrate (%),

18.43±0.80, 19.08±0.74, 18.01±0.24, 39.83±0.38, 39.35±0.66, 39.18±0.83,

40.07±0.66, 40.16±0.24, 39.59±0.78; organic matter (%), 95.64±0.03, 95.72±0.02,

95.95±0.03, 96.15±0.08, 96.02±0.69, 96.34±0.20, 94.52±0.24, 94.69±0.17,

94.63±0.36; total dietary fiber (%), 5.69±0.11, 5.73±0.19, 5.62±0.17, 11.91±0.21,

11.79±0.19, 12.09±0.27, 14.12±0.11, 14.31±0.31, 14.32±0.27; total free amino acids

(% E of Leucine), 0.034±0.0, 0.035±0.0, 0.036±0.001, 0.056±0.001, 0.057±0.001,

0.057±0.001, 0.044±0.001, 0.045±0.001, 0.045±0.002; gross energy (Kcal/100g

LPC), 472.39±5.13, 469.24±1.87, 475.18±0.58, 447.29±1.26, 447.71±3.30,

449.77±3.40, 463.88±1.51, 463.61±1.34, 464.97±2.70; pigments (chlorophyll (mg/g

LPC), 4.64±0.02, 4.68±0.01, 4.65±0.04, 3.77±0.07, 3.70±0.06, 3.69±0.09, 5.18±0.07,

5.25±0.08, 5.20±0.01; total carotenes (mg/100g LPC), 19.94±0.66, 19.91±0.39,

19.99±0.34, 25.29±0.32, 25.22±0.78, 25.02±0.36, 27.44±0.16, 27.38±0.07,

27.41±0.17; xanthophylls (%), 0.21±0.04, 0.21±0.03, 0.22±0.01, 0.82±0.01,

0.83±0.03, 0.83±0.02, 0.54±0.01, 0.54±0.02, 0.54±0.01); minerals (mg/100g LPC),

sodium, 5.11±0.25, 5.06±0.17, 5.17±0.08, 4.72±0.17, 4.56±0.19, 4.42±0.47,

14.16±0.15, 14.23±0.08, 14.26±1.02; calcium, 1976.3±40.51, 1971.3±39.37,

1988.7±61.99, 1079±18.72, 1087±23.02, 1070±12.12, 2478.7±71.30, 2530±122.39,

2453±84.26; phosphorus, 576±25.24, 570.33±18.15, 583±39.95, 746.33±10.21,

747.33±21.50, 754±13, 620.67±2.08, 616±6.25, 618.33±9.45; iron, 70.47±1.02,

70.44±1.57, 70.48±1.51, 53.20±0.49, 53.42±0.29, 55.05±1.31, 65.48±0.69,

65.42±1.39, 63.63±1.41; copper, 17.14±0.36, 16.82±0.22, 16.92±0.04, 13.12±0.17,

13.13±0.18, 12.95±0.23, 8.09±0.20, 7.62±0.31, 7.48±0.25; zinc, 9.38±0.16,

9.16±0.14, 9.18±0.10, 7.57±0.25, 7.84±0.09, 8.05±0.23, 4.11±0.05, 4.19±0.15,

3.87±0.19; manganese, 0.28±0.0, 0.26±0.01, 0.28±0.01, 0.38±0.01, 0.37±0.01,

0.38±0.01, 0.33±0.01, 0.33±0.01, 0.32±0.02; magnesium, 21.65±0.11, 20.78±0.38,

20.84±0.11, 27.66±0.35, 26.62±0.80, 27.32±0.30, 21.66±0.47, 21.55±0.41,

20.27±0.73; and potassium, 17.58±0.33, 17.50±0.40, 16.76±1.07, 21±0.19,

20.68±0.28, 21.05±0.08, 14.64±0.30, 15.09±0.26, 14.59±0.55; respectively. The in

vitro protein digestibility (%) determined in PDLPCL, PDLPCM, PDLPCU,

PPLPCL, PPLPCM, PPLPCU, ALLPCL, ALLPCM, and ALLPCU in both Pepsin-

190

Pancreatin and Pepsin- Trypsin was 86.40 ±0.53, 86.45 ±0.99, 86.60 ±0.96, 82.72

±0.30, 82.83 ±0.26, 82.62 ±0.09, 77.02 ±0.81, 76.89 ±0.30, 77.26 ±0.71 and 86.00

±0.37, 86.32 ±0.62, 86.47 ±0.96, 82.95 ±0.19, 82.83 ±0.24, 82.81 ±0.43, 76.78 ±0.68,

76.86 ±0.69, 76.99 ±0.57, respectively. Anti-nutritional factors such as total

polyphenols (% GAE) in PDLPCL, PDLPCM, PDLPCU, PPLPCL, PPLPCM,

PPLPCU, ALLPCL, ALLPCM, and ALLPCU was 0.084±0.001, 0.084±0.001,

0.085±0.001, 0.122±0.006, 0.123±0.006, 0.120±0.004, 0.032±0.001, 0.032±0.001 and

0.031±0.001 % GAE, respectively. Saponins were not detected in the LPCs isolated

from PDLPC and ALLPC. Total saponins content determined in the PPLPCs

(PPLPCL, PPLPCM, PPLPCU) was 0.51±0.01, 0.51±0.02 and 0.50±0.01%,

respectively. Total alkaloids were found to be absent in all the LPCs examined.

Statistical analysis of these data revealed that the canopy of the trees did not have

significant influence on the yield and nutritional characteristics of these LPCs

(p>0.05). However, PDLPC, PPLPC and ALLPC when compared together

irrespective of canopy showed significant difference in their yield and biochemical

composition. The proximate nutritional composition, in vitro digestibility and anti

nutritional factors determined in these LPCs when compared to those reported in

literature for the LPCs recovered from several plant species including trees

(leguminous and non leguminous), herbs (leguminous and non leguminous), grasses,

aquatic weeds, cultivated crops, were found to be in good agreement, and suitability

of these LPCs as a good protein source was, therefore, inferred.

It was demonstrated that the biomass residue (leaves) generated from the

Populus deltoides, Pongamia pinnata and Albizia lebbeck plantations could be

utilized for development of LPC as a food/ feed supplement to combat protein

deficiency. Further, possibility of the utilization of the by products – residual fiber

and the residual liquid (whey) generated during production of these LPCs for varied

applications such as (i) to make biogas, which in turn can be used to heat the leaf juice

(ii) as a substrate to grow mushrooms (iii) as a soil enriching organic mulch and or

fertilizer for intensive vegetable gardening (iv) to make paper and fiber board (v) to

feed the ruminant animals (vi) as a good culture medium for microorganism (vii) a

soil amender and (viii) an additive, after evaporative down to a syrupy consistency, to

feed for sale to animal feeding operations was also explored.

Thus, a new chemical utilization approach of the leaves, the underutilized

biomass residues from Populus deltoides, Pongamia pinnata and Albizia lebbeck

191

plantations was shown. This is in tune with the emerging biorefinery concept which

has been attracting increasing interest in recent years from the perspective of

upgrading the agro forest biomass residues for value added utilization. The study is

of immense benefits to the farmers in getting another income from these plantations.

This is the first report on the isolation and biochemical composition assessment of the

LPCs from Populus deltoides, Pongamia pinnata and Albizia lebbeck. However,

further research towards in vivo evaluation and amino acids composition of these

LPCs for their quality assessment and commercial utilization are needed to be carried

out.

192

LIST OF PUBLICATIONS

1. Lutful Haque Khan, V.K.Varshney and Sanjay Naithani (2014). Utilization of

biomass residue (leaves) generated from Populus deltoides plantations for

development of protein concentrate. Waste & Biomass Valorization. DOI

10.1007/s12649-014-9306-7.

2. Lutful Haque Khan, V.K.Varshney (2014). Optimization of process conditions

for recovery of leaf protein concentrate from Pongamia pinnata and its

proximate nutritional composition. Industrial Crops and Products.

(Communicated).

3. Lutful Haque Khan, V.K.Varshney (2014). Chemical utilization of Albizia

lebbeck leaves for developing protein concentrates as a nutritional supplement.

Food Science and Biotechnology. (Communicated).

193

PAPERS PRESENTED IN THE CONFERENCE/ SYMPOSIA

1. Standardization of conditions for isolation of leaf protein concentrates from

Pongamia pinnata. In: Symposium on ‘Advances in natural products

chemistry and sustainable development’ held at Graphic Era University,

Dehradun on June 04-05, 2011. (Awarded consolation prize).

2. Utilization of the leaves from Populus deltoides for production of protein

concentrates. In: 6th UCOST, held at Kumaun University, Almora from

November 14-16, 2011.

3. Optimization of extraction procedure for leaf protein concentrate from

Pongamia pinnata and Populus deltoides.In: ‘1st Indian Forest Congress

2011’, held in New Delhi from 22nd -25th November, 2011.

4. Utilization of the leaves from Pongamia pinnata for production of protein

concentrates. In: ‘Green Chemistry for environments and sustainable

development’ by UGC-SAP Department of Chemistry, HNB Garhwal

University held at Dehradun from 11th -13th March, 2012.

5. Chemical utilization of Populus deltoides for developing leaf protein

concentrate. In: 24th Session of International Poplar Commission, held at

Forest Research Institute, Dehradun on 30thOctober -2nd November, 2012.

6. Isolation and nutritional evaluation of leaf protein concentrate from Pongamia

pinnata. In: 7th UCOST, held at Graphic Era University, Dehradun on

November 21-23, 2012.

7. Isolation and nutritive potential of Albizia lebbeck leaf protein concentrate. In:

8th UCOST, held at Doon University, Dehradun on December 26-28, 2013.

1 23

Waste and Biomass Valorization ISSN 1877-2641 Waste Biomass ValorDOI 10.1007/s12649-014-9306-7

Utilization of Biomass Residue (Leaves)Generated from Populus deltoidesPlantations for Development of ProteinConcentrate

Lutful Haque Khan, V. K. Varshney &Sanjay Naithani

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media Dordrecht. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

ORIGINAL PAPER

Utilization of Biomass Residue (Leaves) Generated from Populusdeltoides Plantations for Development of Protein Concentrate

Lutful Haque Khan • V. K. Varshney •

Sanjay Naithani

Received: 12 January 2014 / Accepted: 7 April 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Substantial amount of the leaves as biomass

residue is produced from Populus deltoides (poplar) plan-

tations which has no commercial value and remain un-

derutilized. Feasibility of utilization of this biomass residue

for development of protein concentrate as a food/feed

supplement was examined. A process for isolation of leaf

protein concentrate (LPC) was optimized through investi-

gating the influence of various process parameters on the

yield and protein content of the LPC. The parameters

optimized were ratio of fresh leaves to water (1:9), coag-

ulation temperature (90 �C) and duration (11 min), and pH

(4.0). The optimized process was applied for isolation of

LPCs from lower, middle and upper canopy of the tree, and

the LPCs containing protein 59.24 ± 0.50, 59.56 ± 0.40

and 59.66 ± 0.10 % were recovered in 7.79 ± 0.01,

7.66 ± 0.12 and 7.83 ± 0.14 % yield, respectively. The

proximate nutritional composition (moisture, 6.91, 7.08,

7.08 %; fat, 17.96, 16.95, 18.27 %; crude fibre, 1.54, 1.53,

1.54 %; ash, 4.35, 4.38, 4.36 %; carbohydrates, 18.43,

19.08, 18.01 %; organic matter, 95.64, 95.72, 95.95 %;

total free amino acids, 0.034, 0.035, 0.036 % E of Leucine;

pigments (chlorophyll, 4.64, 4.68, 4.65 mg/g; total caro-

tene 19.94, 19.91, 19.99 mg/100 g; xanthophylls, 0.21,

0.21, 0.22 %); minerals (Na, 5.11, 5.06, 5.17 mg/100 g;

Ca, 1.98, 1.97, 1.99 %; P, 0.58, 0.57, 0.58 %; Fe, 70.47,

70.44, 70.48 mg/100 g), in vitro digestibility (86.20, 86.39,

86.54 %) and anti-nutritional factors (total polyphenols,

0.084, 0.084, 0.085 % gallic acid equivalent) of these LPCs

were determined. Analysis of variance of these data

revealed no significant difference with respect to canopy. It

was inferred that the poplar leaves hold potential for

development of LPC as a food/feed supplement to combat

nutrition deficiency.

Keywords Leaf protein concentrate � Populus deltoides �Nutritional profile � Anti-nutritional factors � Digestibility

Introduction

With rapid population growth coupled with limited culti-

vable land, shortage of protein has become a serious

problem in developing world. Consequently, malnutrition

caused by protein deficiency is wide spread in these

countries [1]. Further, shortage of supply of good quality

protein is also a serious concern of the feed producers to

produce animal protein. Use of human dietary and animal

protein such as soybean and fish in livestock and fish diets,

particularly in fish meals, is unaffordable in the developing

economies. This limits aquaculture development since fish

requires more protein than terrestrial animals [2]. These

considerations have necessitated the search for additional

sources of protein. Several novel sources of protein such as

fish protein concentrate [3], single cell protein [4], soybean

protein [5] and insect protein [6] have been suggested to

meet the increasing demand of human dietary and animal

protein.

Leaf protein concentrate (LPC), a concentrated form of

protein derived from the foliage of plants, has long been

recognised [7] as the inexpensive and most abundant

potential source of proteins for production of animal feed

and unconventional foods because of their capacity to

L. H. Khan � V. K. Varshney (&)

Chemistry Division, Forest Research Institute,

Dehra Dun 248006, India

e-mail: varshney2000@yahoo.com

S. Naithani

Chemistry Division, Institute of Wood Science and Technology,

Bangalore 560003, India

123

Waste Biomass Valor

DOI 10.1007/s12649-014-9306-7

Author's personal copy

synthesize amino acids from a wide range of virtually

unlimited and readily available primary materials such as

sunlight, water, CO2 and atmospheric N2 [8]. Nutritional

value of LPC extracted from green foliage has been shown

to be comparable to that of protein isolates of animal origin

and superior to seed proteins [9]. LPCs have been assessed

as food for children [10] and included in food formulations

[11]. Use of LPCs as a protein supplement in animal feed

has also been demonstrated [12, 13]. In recognition of the

viable role of LPCs in combating protein deficiency, sev-

eral studies aimed at production of protein concentrates

from green leaf material, their characterization in terms of

nutritional and physico-chemical attributes, and utilization

as quality food and feed have been carried out [14–18].

Trees have also been suggested as a possible source of

LPC and the production of protein from tree leaves is

advantageous over crops as they do not involve recurring

cost of cultivation [18, 19]. Populus deltiodes (commonly

known as poplar), being fast grown, easy to propagate and

highly adaptable to different climatic and soil conditions, is

planted on a large scale in India. Substantial amount of

leaves as a biomass residue is produced from poplar

plantations during harvesting of wood for commercial

applications. This biomass residue has no commercial

value and is currently burned or underutilized. Burning of

these leaves can results in the release of green house gases

into the atmosphere. Thus, poplar leaves may have serious

environmental impact from their disposal. This has

necessitated effects to find ways of reducing their envi-

ronmental impact or transferring them into value-added

products.

Prompted by above facts, feasibility of utilization of

poplar leaves for development of protein concentrates as a

food/feed supplement was examined and results of the

study are reported herein. To the best of our knowledge,

there is no work investigating the potential of poplar leaves

for production of LPC.

Materials and Methods

Plant Materials

Fresh leaves of Populus deltoides were collected in the

early morning from the trees grown in the campus of Forest

Research Institute, Dehradun, India which lies between

latitudes 29�580N and 31�20N and longitudes 77�340E and

78�180E. The plant material was authenticated at System-

atic Botany Discipline, Botany Division, Forest Research

Institute, Dehradun, India. The leaves were analyzed for

moisture content using oven dry procedure. Based on the

moisture content (65.01 ± 0.42 %), the average dry matter

content of the leaves was 34.99 ± 0.42 %.

Chemicals and Reagents

Citric acid, Potassium sulphate, Copper sulphate, Sulphuric

acid, Bromocresol green, Methyl red, Ortho phosphoric

acid, Diphenyl amine indicator, Ferrous ammonium sul-

phate, Ninhydrin, Stannous chloride, Sodium citrate,

n-Propanol, Potassium hydroxide, Hexane, Toluene,

Ammonium oxalate, Potassium permanganate, Ammonium

molybdate, Potassium nitrate, Folin-Ciocalteu’s Phenol

reagent, Sodium carbonate and Sodium azide were pur-

chased from Merck India. Acetic acid, Hydrochloric acid,

Sodium Hydroxide, Boric acid, Petroleum ether, Acetone,

Sodium sulphate, Ammonia solution, Nitric acid, Chloro-

form and Ethyl acetate were from Rankem, India. Potas-

sium dichromate, Methoxy ethanol, Methanol, Diethyl

ether and Ethanol were supplied by Sd Fine Chem. Ltd.

India. Dimethyl sulfoxide, Phenolphthalein were procured

from Ranbaxy, India. Leucine, Pepsin, Trypsin, and Pan-

creatin were obtained from Himedia, India and Gallic acid

from Sigma Chemicals Co. (St. Louis, MO, USA).

Isolation of Leaf Protein Concentrate from Populus

deltoides (PDLPC)

Fresh leaves (100 g) were washed with tap water, drained

and minced with distilled water (1:1–1:11, w/v) in a locally

purchased mixer followed by filtration through muslin

cloth. The leaf protein from the separated juice was

coagulated by 1N acetic acid at ambient temperature or by

heating after adjusting the pH (acidic pH-4.0, basic pH-9.0

and bio pH-7.0) for the desired temperature (50–100 �C)

for fixed duration (1–15 min). The coagulum was centri-

fuged at 10,000 rpm for 10 min, washed with distilled

water till neutral pH and oven dried until constant weight.

Isolation of LPCs from Lower, Middle and Upper

Canopy of Populus deltoides

The LPCs were isolated from lower (PDLPCL), middle

(PDLPCM) and upper canopy (PDLPCU), of Populus

deltoids using the optimized protocol.

Determination of Proximate Nutritional Composition

The LPCs (PDLPCL, PDLPCM, PDLPCU) were analyzed

for the content of moisture, nitrogen, organic matter, protein

(N X 6.25), fat, crude fiber, ash, total carbohydrates, total

free amino acids (TFAAs), minerals (Ca, P, Na and Fe),

pigments (chlorophylls, total carotenes and xanthophylls),

in vitro protein digestibility and anti-nutritional factors (total

polyphenols, total saponins and total alkaloids) using the

methods of Association of Official Analytical Chemists

(AOAC, 1975 and 1995) and published in literature [20–24].

Waste Biomass Valor

123

Author's personal copy

Statistical Analysis

The experiments were done in triplicate and the values

determined are presented as mean ± standard deviation.

The data were subjected to one-way analysis of variance

(ANOVA) using the SPSS version 16.00 (Statistical

Package for Social Sciences Version 16.00). Statistical

significance of the differences was performed by post hoc

analysis using Tukey test. Differences with p \ 0.05 were

considered significant.

Results and Discussion

The procedure for isolation of PDLPC was optimized with

respect to the yield of LPC (expressed in g/100 g leaves)

and protein content (%) by varying the process parameters

as shown in Table 1. Each of these parameters was varied

one by one keeping the remaining parameters constant. The

yield and the protein content of the LPCs obtained under

varied process conditions were determined and their

dependence on each of the variables was investigated.

The effect of the ratio of fresh leaves to water on the

yield of LPC and protein content was studied as shown in

Fig. 1. It was observed that the LPC yield and protein

content increased from 2.77 to 7.96 g and 43.18 to

58.95 %, respectively with increasing the ratio of fresh

leaves to water from 1:1 to 1:9, and thereafter remained

constant. The increase in the LPC yield and protein content

up to 7.96 g and 58.95 %, respectively could be attributed

to good flowability of the leaf slurry and softening of the

cell wall resulted from the increased moisture content [17].

The influence of the fresh leaves to water ratio on LPC

yield and protein content was found to be statistically

significant (p \ 0.05).

With the ratio of fresh leaves to water 1:9, the effect of

pH required to cause protein coagulation, on the LPC yield

and protein content was examined. For this, protein was

coagulated using 1N acetic acid at room temperature, and

at different pH viz, acidic pH-4.0 (adjusted by 1 N HCl),

basic pH-9.0 (adjusted by 1N NaOH) and bio pH-7.0 fol-

lowed by heating in boiling water. The results are pre-

sented in Fig. 2. There was significant difference in the

yield of LPC and protein content under above varied

conditions. Use of 1N HCl followed by heating in boiling

water produced LPC with the highest yield (7.91 g) and

protein content (59.38 %).

Coagulating the protein at pH-4.0 using 1N HCl, effect

of coagulation temperature varied from 50–100 �C, on the

yield of LPC and protein content was investigated and the

results are illustrated in Fig. 3. On increasing the temper-

ature from 50 to 90 �C, LPC yield and protein content were

found to increase from 3.13 to 7.95 g and 44.66 to

59.44 %, respectively. However, further increase in tem-

perature to 100 �C resulted in no appreciable change in the

LPC yield and the protein content. This may probably be

attributed to the non availability of the enough protein for

Table 1 Process conditions for isolation of LPC from P. deltoides

Leaves: water ratio (w/v) 1:1–1:11

Coagulation temperature (�C) 50–100

Coagulation duration (min) 1–15

Coagulation factor pH-4.0 and heating at 100 �C

pH-9.0 and heating at 100 �C

Bio-pH and heating at 100 �C

1N Acetic acid coagulation at

ambient temperature

Optimization of different

acids to bring pH-4.0

1N Acetic acid, 1N citric acid

and 1N HCl

2.77 4.99 5.48 6.37 7.96 7.92

43.1846.35

49.4954.01

58.95 59.12

0

10

20

30

40

50

60

70

01:01 01:03 01:05 01:07 01:09 01:11

Yie

ld o

f L

PC (

g) &

Pro

tein

(%

)

Fresh leaves to water ratio (w/v)

LPC

Protein

Fig. 1 Effect of fresh leaves to

water (w/v) ratio on the yield of

LPC and corresponding protein

content

Waste Biomass Valor

123

Author's personal copy

coagulation. The increment in yield and protein content

with increasing the temperature up to 90 �C could be due to

the favorable effect of temperature on protein coagulation.

Coagulation of the chloroplast protein has been reported up

to the temperature of 70 �C beyond which cytoplasm

protein is coagulated [7]. Thus the coagulation temperature

was optimized to 90 �C. Above observation was also ver-

ified from the statistical analysis of the data which were

found to be statistically significant at the temperature from

50 to 90 �C and statistically insignificant at temperature

from 90 to 100 �C.

Using the above optimized parameters (leaves to water

ratio 1:9, coagulation pH-4.0 and temperature 90 �C), the

effect of coagulation duration varied from 1 to 15 min, was

studied on the LPC yield and protein content. As shown in

Fig. 4, an increase in the LPC yield (2.73–7.96 g) and

protein content (23.62–59.48 %) was observed with the

increase in duration from 1 to 11 min and thereafter, the

yield of LPC and protein content remained almost constant.

Statistical analysis of the data also confirmed the above

findings. Thus the coagulation duration was optimized to

11 min.

Experiments were also carried out to bring the pH of the

juice to 4 using 1N acetic acid (72 ml), 1N HCl (8 ml) and

1N citric acid (19 ml) before heating it in boiling water.

There was no significant difference in the yield of LPC and

protein content ranged from 7.82–7.99 g and

59.14–59.20 %, respectively. These three acids followed

the order, in terms of the quantity required to coagulate

protein at pH-4.0, Acetic acid [ Citric acid [ HCl.

Although the quantity of 1N HCl was the lowest, appli-

cation of the citric acid was found to be suitable in view of

the environmental concerns associated with the use of HCl.

Based on the above experiments, the optimized protocol

and set of conditions for isolation of LPC from P. delto-

ides, is illustrated in Fig. 5.

Using the standard process conditions, PDLPCL,

PDLPCM, PDLPCU were isolated and yield was deter-

mined to be 7.79 ± 0.01, 7.66 ± 0.12 and 7.83 ± 0.14 g,

respectively which was statistically insignificant (p [ 0.05).

5.71 6.39 7.11 7.91

53.17 55.33 56.79 59.38

0

10

20

30

40

50

60

70

Bio-pH &Heat at 100°C

Basic pH (9.0)& Heat at

100°C

AcidicCoagulation

Acidic pH(4.0) & Heat at

100°CY

ield

of

LPC

(g)

& P

rote

in (

%)

Different coagulation parameters

LPC

Protein

Fig. 2 Effect of different

coagulation parameters on the

yield of LPC and corresponding

protein content

3.13 4.72 5.85 6.75 7.95 7.92

44.66 46.55

53.8455.33

59.44 59.25

0

10

20

30

40

50

60

70

50°C 60°C 70°C 80°C 90°C 100°C

Yie

ld o

f L

PC (

g) &

Pro

tein

(%

)

Temperature (°C)

LPC

Protein

Fig. 3 Effect of coagulation

temperature on the yield of LPC

and corresponding protein

content

Waste Biomass Valor

123

Author's personal copy

Proximate Nutritional Composition

The proximate nutritional composition of PDLPCL,

PDLPCM, and PDLPCU is given in Table 2. The observed

moisture content of PDLPCL, PDLPCM and PDLPCU was

6.91 ± 0.19, 7.08 ± 0.20 and 7.08 ± 0.08 %, respec-

tively. Moisture in food determines the rate of food

absorption and assimilation within the body. Moisture

content of LPC also determines its keeping quality and

\10 % moisture content is recommended [18]. LPCs

extracted from Moringa oleifera, Telfeira occidentalis and

Amarathus hybridus have been reported to contain mois-

ture content in the range 6.6–9.0 % [14, 18]. Our findings

are in good agreement with the reported ones.

PDLPCL, PDLPCM and PDLPCU contained apprecia-

ble amount of nitrogen (9.47 ± 0.08, 9.53 ± 0.06 and

9.54 ± 0.03, respectively) and crude protein

(59.24 ± 0.50, 59.56 ± 0.40 and 59.66 ± 0.10,

2.73 4.2 5.12 5.97 6.5 7.96 7.96 7.99

23.62

33.337.47

48.6653.57

59.48 59.36 59.48

0

10

20

30

40

50

60

70

1 3 5 7 9 11 13 15

Yie

ld o

f L

PC (

g) &

Pro

tein

(%

)

Coagulation Time (min)

LPC

Protein

Fig. 4 Effect of coagulation

duration on the yield of LPC

and corresponding protein

content

Fresh leaves

Pulping and mincing with distilled water (1:9, w/v)

Fibrous residue

Leaf juice

Adjust the pH of the leaf juice to pH-4.0 using 1N Citric acid and heat thejuice at water bath temperature of 90 °C for 11 minutes.

Cool the extract at room temperature

Centrifuge the extract

Whey fraction

Wash the residue with distilled water using a vacuum pump

Drying

LPC (Dry Matter)

Fig. 5 Optimized protocol and

set of conditions for isolation of

LPC from P. deltoides

Waste Biomass Valor

123

Author's personal copy

respectively). It is generally recommended that a leaf

suitable for the preparation of LPC should contain at least

20 % protein [25]. Further, for human consumption and

animal feeding, LPCs containing protein content 55–65 %

and 55 %, respectively have been found suitable [26]. The

proteins taken in as food are indispensable to ensure the

renewal of the amino-acids required for the synthesis of the

structural and functional cells of the body. The proportion

of crude protein content, irrespective to canopy, is com-

parable to that reported for Lucerne LPC (50–60 % crude

protein) which is produced at the industrial scale. The

recommended dietary allowance of protein for children,

adult males and females are 28, 63 and 50 gm, respectively

[27]. The aforesaid facts indicate that PDLPC is a good

source for daily proteins.

The fats are main source of energy in body but should

not exceed the daily recommended dose of not more than

30 calories [28]. Besides they are also involved in the

making of hormones, prostaglandins and are also necessary

for the absorption of some fat soluble vitamins (A, D, E

and K), permeability of cell membranes, the transmission

of nerve impulses, the metabolism of adrenal hormones and

male fertility [27]. They are, therefore, of great importance

physiologically, both to animals and man. The fat contents

in PDLPCL, PDLPCM and PDLPCU were 17.96 ± 1.02,

16.95 ± 0.37 and 18.27 ± 0.09 %, respectively. This

value is much [9.20 % for Vernonia amygdalina [18],

0.3 % for spinach leaves, 0.4 % for chaya leaves, 1.60 %

in Amaranthus hybrids leaves [29] and 8–12 % for Medi-

cago sativa [27].

Crude fiber contents of PDLPCL, PDLPCM and

PDLPCU were determined to be 1.54 ± 0.07, 1.53 ± 0.05

and 1.54 ± 0.03 %, respectively. These contents are lower

than those reported in V. amygdalina leaf concentrate

(10.46 %), sweet potato protein concentrate (7.20 %) [18]

but comparable to Lucerne LPC (\2 %) [27]. The low fibre

level both allows the concentration of useful components

such as minerals and improves their assimilation in the

digestive tract. The fibre is also necessary for the good

functioning of the bowel [27].

Ash content is a measure of minerals found in the food.

The ash contents of PDLPCL, PDLPCM and PDLPCU were

found to be 4.35 ± 0.03, 4.38 ± 0.02 and 4.36 ± 0.02 %,

respectively. For the use as food and feed, the recom-

mended ash content of the LPC are 2–5 % and 11 %,

respectively [26]. Our findings in are in accordance to the

reported observations.

Carbohydrates content of PDLPCL, PDLPCM and

PDLPCU were 18.43 ± 0.80, 19.08 ± 0.74 and

18.01 ± 0.24 %, respectively. Carbohydrates are the

body’s principle and most economical energy source.

Carbohydrate is widely available in normal diets but the

preferred sources are cereals and vegetables. There is no

report on daily requirements of carbohydrate but 50 gm of

carbohydrates should be enough to prevent breakdown of

the body’s store of protein and fats [18]. Lucerne LPC

contains 6 % sugars and the recommended dosage of LPC

is 6–15 g/day which can help to stabilize glycaemia [27].

Our findings show that PDLPC is a good source of

energy.

Table 2 Proximate nutritional

composition of the LPCs

isolated from lower, middle and

upper canopy of P. deltoides

*The values are mean of 3

replicates ± SD; ** NS not

significant at p [ 0.05

Nutritional parameters PDLPCL* PDLPCM* PDLPCU* Significance

level**

Moisture (%) 6.91 ± 0.19 7.08 ± 0.20 7.08 ± 0.08 NS

Protein (%) 59.24 ± 0.50 59.56 ± 0.40 59.66 ± 0.10 NS

Fat (%) 17.96 ± 1.02 16.95 ± 0.37 18.27 ± 0.09 NS

Crude fibre (%) 1.54 ± 0.07 1.53 ± 0.05 1.54 ± 0.03 NS

Ash (%) 4.35 ± 0.03 4.38 ± 0.02 4.36 ± 0.02 NS

Total carbohydrates (%) 18.43 ± 0.80 19.08 ± 0.74 18.01 ± 0.24 NS

Organic matter (%) 95.64 ± 0.03 95.72 ± 0.02 95.95 ± 0.03 NS

Total free amino acids (% E of Leucine) 0.034 ± 0.0 0.035 ± 0.0 0.036 ± 0.001 NS

Pigments

Chlorophyll (mg/g LPC) 4.64 ± 0.02 4.68 ± 0.01 4.65 ± 0.04 NS

Carotene (mg/100 g LPC) 19.94 ± 0.66 19.91 ± 0.39 19.99 ± 0.34 NS

Xanthophylls (%) 0.21 ± 0.04 0.21 ± 0.03 0.22 ± 0.01 NS

Minerals

Sodium (mg/100 g LPC) 5.11 ± 0.25 5.06 ± 0.17 5.17 ± 0.08 NS

Calcium (%) 1.98 ± 0.04 1.97 ± 0.04 1.99 ± 0.06 NS

Phosphorus (%) 0.58 ± 0.03 0.57 ± 0.02 0.58 ± 0.04 NS

Iron (mg/100 g LPC) 70.47 ± 1.02 70.44 ± 1.57 70.48 ± 1.51 NS

Waste Biomass Valor

123

Author's personal copy

PDLPCL, PDLPCM and PDLPCU were found to con-

tain very good amount of organic matter (95.64 ± 0.03,

95.72 ± 0.02 and 95.95 ± 0.03 %), respectively. It is very

important in the movement of nutrients and plays a role in

water retention. Total free amino acids contents of

PDLPCL, PDLPCM and PDLPCU were determined to be

0.034 ± 0.0, 0.035 ± 0.0 and 0.036 ± 0.001 % Equiva-

lence of Leucine, respectively. These low values are

indicative of the healthy status of the PD leaves, used for

recovery of LPC as the production of the TFAAs is asso-

ciated with the physiological changes resulted from the

disease in the leaves [22].

No significant difference in the contents of moisture,

protein, fat, ash, crude fiber, total carbohydrates, organic

matter and TFAAs determined in the PDLPCL, PDLPCM

and PDLPCU, as revealed by ANOVA analysis of these

data, was observed.

Pigments

The concentration of pigments such as chlorophyll, caro-

tene and xanthophylls, determined in PDLPCL, PDLPCM

and PDLPCU, is presented in Table 2. Chlorophyll con-

tents were determined to be 4.64 ± 0.02, 4.68 ± 0.01 and

4.65 ± 0.04 mg/g, respectively. Chlorophyll estimation is

one of the important biochemical parameters which is used

as the index of production capacity. Also chlorophyll is

very much similar to haemoglobin. The human body has

the ability to convert chlorophyll to haemoglobin by

changing magnesium to iron. This allows chlorophyll to be

quickly absorbed by the human body and functions as a

good blood builder to rebuild the blood stream. While

promoting and stimulating the availability of red blood

cells to the body it also increases function of the heart,

lungs, and vascular system. This can also help the immune

system because it improves the circulation and oxygenation

of the cells and body [27].

Total carotene, another important component of leaf

protein, is also nutritionally important biochemical param-

eters. As shown in Table 2, the PDLPCL, PDLPCM and

PDLPCU contained total carotene content 19.94 ± 0.66,

19.91 ± 0.39 and 19.99 ± 0.34 mg/100 g LPC, respec-

tively. Amongst the carotenes, the most important one is b-

carotene which is important in lessening asthma symptoms,

preventing certain cancers, heart disease, cataracts and age

related macular degeneration and treating AIDS, alcohol-

ism, Alzheimer’s disease, Parkinson’s disease and

skin disorders such as psoriasis. b-carotene is also used in

malnourished (underfed) women to reduce the chance of

death and night blindness during pregnancy as well as

diarrhea and fever after giving birth [27]. The recom-

mended dose of b-carotene for children is 0.38 mg per kg

body weight [30].

Xanthophylls concentration in PDLPCL, PDLPCM and

PDLPCU was 0.21 ± 0.04, 0.21 ± 0.03 and 0.22 ±

0.01 %, respectively. In recent years, a great deal of

attention has been focused on biological activities of die-

tary xanthophylls such as lutein, zeaxanthin, b-cryptoxan-

thin, capsanthin, astaxanthin, and fucoxanthin [31].

Xanthophylls have been thought to work as antioxidants

[32] and as blue light filters [33] to protect the eyes from

oxidative stresses. Numerous studies have reported that

Xanthophylls has the potential to prevent cancers, diabetes,

and inflammatory and cardiovascular diseases [34].

There was no significant difference in the content of

chlorophyll, total carotene and xanthophylls of the

PDLPCL, PDLPCM and PDLPCU, as revealed by ANOVA

analysis of these data (p [ 0.05). It was inferred that

PDLPC irrespective of the canopy can be a good source of

pigments (chlorophyll, total carotene and xanthophylls).

Minerals

Table 2 presents the mineral (Na, Ca, P, Fe) estimated in

PDLPCL, PDLPCM and PDLPCU. These are largely water

soluble. They are active in many reactions and in tissue

building. The role of each element is not yet very well defined:

some are merely catalysts, others have both plastic (tissue

structure) and catalytic functions. It must be said, too, that

some plant acids form with minerals insoluble salts which

makes them unavailable (e.g. oxalic acid in some leaves and

phytic acid in cereals). They are not synthesised by the body

and so must be taken in food [27]. Sodium concentration in the

PDLPCL, PDLPCM and PDLPCU was 5.11 ± 0.25,

5.06 ± 0.17 and 5.17 ± 0.08 mg/100 g LPC, respectively

and was statistically insignificant (p [ 0.05). Sodium is the

principal extracellular cation in the body. It also acts as an

antagonistic and complementary to potassium. The daily

allowance of sodium is 500 mg for adult [25]. Calcium is

required by children, pregnant and lactating woman for for-

mation and growth of bones and teeth. It also acts as a catalyst

in blood coagulation in the body. The concentration of cal-

cium in the PDLPCL, PDLPCM and PDLPCU was

1.98 ± 0.04, 1.97 ± 0.04 and 1.99 ± 0.06 %, respectively.

The recommended daily allowance of calcium is

600–700 mg, 900 mg, and 1 200 mg for child (1–9 years),

adult and adolescent, elderly, lactating women, respectively

[27]. This indicates that PDLPC can be a good contributor of

calcium. Phosphorus is another important mineral required by

children, pregnant and lactating woman for formation and

growth of bones, teeth and cell membranes. It is also a key

player in fat metabolism. It also acts as a catalyst in phos-

phorylation reaction. The concentration of phosphorus in the

PDLPCL, PDLPCM and PDLPCU was 0.58 ± 0.03,

0.57 ± 0.02 and 0.58 ± 0.04 %, respectively. The

Waste Biomass Valor

123

Author's personal copy

recommended daily allowance of phosphorus is 500–600, 800

and 1 000 mg for child (1–9 years), adult and adolescent,

elderly, lactating women, respectively [27]. This indicates that

PDLPC can be a good source of phosphorus. The iron con-

centration in the PDLPCL, PDLPCM and PDLPCU was

70.47 ± 1.02, 70.44 ± 1.57 and 70.48 ± 1.51 mg/100 g

LPC. Iron is required for haemoglobin synthesis and its defi-

ciency leads to anaemia. Iron is anti-infective and also

essential ion for respiration. The daily requirement of iron is

10, 10–18 and 10–18 mg for child (1–9 years), adult and

adolescent, elderly, lactating women, respectively [27]. These

reported values of iron indicate that PDLPC can provide the

daily requirement of iron.

In Vitro Protein Digestibility

In vitro protein digestibility of PDLPCL, PDLPCM and

PDLPCU was determined using the Pepsin-Pancreatin and

Pepsin- Trypsin methods and the results are shown in

Table 3. The protein digestibility for PDLPCL, PDLPCM

and PDLPCU, determined using both the methods was

[86 % and statistically insignificant (p [ 0.05). The actual

protein digestibility has an important effect on its overall

nutritive value. As pointed out by Sheffner [35], in vitro

methods are useful for nutritive evaluation of protein

quality or digestibility because of their rapidity and sensi-

tivity, but one can only interpret the results with confidence

when they are a complement to in vivo animal tests. The

in vitro digestibility determined here for PD protein con-

centrates are very encouraging. The digestibility of LPC

has been reported to range from 65 to 85 % [23]. The

digestibility values determined for PDLPCL, PDLPCM

and PDLPCU were higher than those reported for wheat

LPC and similar to values found for lupin and rape LPCs

and slightly lower than alfalfa protein concentrates [23].

Thus, PDLPC possesses good in vitro digestibility.

Anti-nutritional Contents

Plant leaves contain some naturally occurring compounds

termed as anti-nutritional factors which limit its biological

utilization on account of their toxicity. It is believed that

most toxic compounds would be removed during the pro-

cessing of LPC [8, 36]. The anti-nutritional contents such

as poly phenols (determined as total phenolic contents

(TPCs) and expressed as % gallic acid equivalent (%

GAE), total saponins and total alkaloids were determined

in PDLPCL, PDLPCM, and PDLPCU, and shown in

Table 3. The TPCs of PDLPCL, PDLPCM and PDLPCU

were 0.084 ± 0.001, 0.084 ± 0.001 and 0.085 ± 0.001 %

GAE, respectively. Statistically, phenolic content in all the

three LPCs were insignificant. Polyphenols are chemically

highly active compounds that can be at the root of several

undesirable effects such as diminution of the absorption of

Fe, oestrogenic effect of coumesterol or of isoflavones and

diminution of protein digestibility [27]. Polyphenols are

well known to react with the e-amino groups of lysine and

form strong covalent bonds [27]. Phenolic content has also

been listed as a factor most often responsible for small

yields of leaf protein [37]. TPCs in LPC from 1.12 to

5.62 % have been reported in Allmania nodiflora and Hy-

grophila spinosa [26]. TPCs in LPC from vegetable and

legume crops were reported to be varied from 1.4 to 2.2 %

[38]. It is also reported that the ratio of TPCs and nitrogen

of LPC has an inverse relationship to protein quality [39].

Thus, the low value of TPCs in PDLPCL, PDLPCM,

PDLPCU are indicative of their good quality. PDLPCL,

PDLPCM and PDLPCU were also examined for total

saponins and total alkaloids contents. These were found to

be absent in these LPCs.

Conclusion

Possibility of utilization of P. deltoides (poplar) plantations

generated biomass residue (leaves) for development of

LPC was investigated. The process parameters standard-

ized for isolation of the LPC consisted of ratio of fresh

leaves to water (1:9), coagulation temperature (90 �C) and

duration (11 min), and pH (4.0). Application of the opti-

mized process enabled to isolate the LPCs containing

protein 59.24 ± 0.50, 59.56 ± 0.40 and 59.66 ± 0.10 %

Table 3 In vitro protein digestibility and anti-nutritional contents of LPCs isolated from lower, middle and upper canopy of P. deltoides

Standard enzymatic system PDLPCL* PDLPCM* PDLPCU* Significance level**

Pepsin-Pancreatin (%) 86.40 ± 0.53 86.45 ± 0.99 86.60 ± 0.96 NS

Pepsin-Trypsin (%) 86 ± 0.37 86.32 ± 0.62 86.47 ± 0.96 NS

Anti-nutritional factors

Total polyphenols contents (% GAE) 0.084 ± 0.001 0.084 ± 0.001 0.085 ± 0.001 NS

Total saponins (%) ND ND ND

Total alkaloids (%) ND ND ND

* The values are mean of 3 replicates ± SD; ** NS not significant at p [ 0.05; ND not detected

Waste Biomass Valor

123

Author's personal copy

in 7.79 ± 0.01, 7.66 ± 0.12 and 7.83 ± 0.14 % yield from

lower, middle and upper canopy of the tree, respectively.

The proximate nutritional composition, in vitro digestibil-

ity and anti nutritional factor determined in these LPCs

indicated their suitability as a good protein source. Based

on statistical analysis of these data, no significant differ-

ence in the yield and nutritional characteristics of the LPCs

isolated from different canopy of the tree was observed. It

was shown that the biomass residue (leaves) generated

from the poplar plantations could be utilized for develop-

ment of LPC as a food/feed supplement. Thus, a new

chemical utilization approach of the poplar leaves, the

underutilized biomass residue from these plantations, was

shown. This is in tune with the emerging biorefinery con-

cept which has been attracting increasing interest in recent

years from the perspective of the upgrading the agro forest

biomass residues for value added utilization. The study was

also found to be beneficial to the farmers in getting another

income from these plantations.

Acknowledgments The authors are thankful to the Director, Forest

Research Institute, Dehradun for encouragement and providing nec-

essary facilities to carry out the work. One of the authors (LHK) is

grateful to the University Grant Commission (UGC), New Delhi,

India, for financial assistance.

References

1. Latharn, M.C.: Human Nutrition in the Developing World. Food

and Agricultural Organization of the United Nations, Rome (1997)

2. Wilson, P.R.: Amino acid nutrition of fish. A new method of

estimating requirement values. In: Collie, M.R., Mcvey, J.P.

(eds.) Proceedings of the U.S.-Japan Aquaculture Nutrition

Symposium, New Port, pp. 28–29. (1991)

3. Pariser, E.R., Wallerstein, M.B., Corkery, C.J., Brown, N.L.: Fish

protein concentrate: panacea for protein malnutrition. In: Inter-

national Nutrition Policy Series (USA), no. 3. Cambridge, Mass,

USA (1978)

4. Ferrianti, M.P., Fiechter, A.: Production and Feeding of Single

Cell Protein. Applied Science Publications, London (1983)

5. Bhatia, J., Greer, F.: Use of soy protein based formulas in infant

feeding. Paediatrics 121, 1062–1068 (2008)

6. Ghaly, A.E., Alkoaik, F.N.: Extraction of protein from common

plant leaves for use as human food. Am. J. Appl. Sci. 7(3), 331–342

(2010)

7. Pirie, N.W.: Leaf Protein: Its Agronomy, Preparation, Quality

and Use. IBP Hand book 20. Blackwell Scientific, Oxford (1971)

8. Ravindrian, V., Blain, R.: Feed resource for poultry production in

Asia and the Pacific II. Plant protein source. World Poult. Sci. 48,

205–231 (1992)

9. Morris,T.R.: Agronomic studies in leaf protein. In: Wilkins, R.J.

(ed.) Green Crop Fractionation. Brit. Grassld. Soc. Occas. Symp.

9 (1977)

10. Kamalanathan, G., Nalinakshi, G.S., Devadas, R.P.: Effect of a

blend of protein foods on the nutritional status of preschool

children in a rural blawadi. Indian J. Nutr. Diet. 7, 288–291 (1970)

11. Maciejewicz-Rys, J., Hanczakowski, P.: Improvement of the

nutritive value of cereals by leaf protein supplementation. J. Sci.

Food Agric. 50, 99–104 (1990)

12. Olvera-Novoa, M.A., Campos, S.G., Sabidoa, M.G., Palacios,

C.A.M.: The use of alfalfa leaf protein concentrate as a protein

source in diets for tilapia (Oreochromis mossambias). Aquacul-

ture 90(3–4), 291–302 (1990)

13. Abowei, J.F.N., Ekubo, A.T.: A review of conventional and

unconventional feeds in fish nutrition. Br. J. Pharmacol. Toxicol.

2(4), 179–191 (2011)

14. Adeyeye, E.I., OmolayO, F.O.: Chemical composition and

functional properties of leaf protein concentrates of Amaranthus

hybridus and Telfairia occidentalis. Agric. Biol. J. N. Am. 2(3),

499–511 (2011)

15. Aletor, O.: Comparative, nutritive and physico-chemical evalu-

ation of Cassava (Mannihot esculanta) leaf protein concentrate

and fish meal. J. Food Agric. Environ. 8(2), 39–43 (2010)

16. Bedor, K.V., Kulkarni, A.U.: LPC is novel source of protein for

human health and nutrition. A review. Curr. Bot. 2, 6–7 (2011)

17. Ghaly, A.E., Mahmond, N., Dave, D.: Nutrient composition of

Dandelious and its potential as human food. Am. J. Biochem.

Biotechnol. 8(2), 118–127 (2012)

18. Sodamode, A., Bolaji, O.S., Adeboye, O.O.: Proximate analysis,

mineral contents and functional properties of Moringa oleifera

leaf protein concentrate. IOSR J. Appl. Chem. 4(6), 47–51 (2013)

19. Pirie, N.W.: Food from the forest. New Sci. 40, 420 (1968)

20. Association of Official Analytical Chemists.: Official Methods of

Analysis of the Association of Official Analytical Chemists. (12th

edn.) Washington, DC, USA (1975)

21. Association of Official Analytical Chemists.: Official Methods of

Analysis of AOAC International. (16th edn.) Washington, DC,

USA (1995)

22. Sadasivam, S., Manickam, A.: Biochemical Methods. New Age

International, New Delhi (2005)

23. Saunders, M.R., Connor, M.A., Booth, N.A., Bickoff, M.E.,

Kohler, O.G.: Digestibility of alfalfa protein concentrates by

in vivo and in vitro methods. J. Nutr. 103, 530–535 (1973)

24. Singleton, V.L., Rossi, J.A.: Colorimetry of total phenolics with

phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol.

Vitic. 16, 144–158 (1965)

25. National Research Council: Recommended Dietary Allowances,

10th edn. The National Academies Press, Washington, DC (1989)

26. Dewanji, A., Chanda, S., Si, S., Barik, S., Matai, S.: Extractability

and nutritional value of leaf protein from tropical aquatic plants.

Plant Food Hum. Nutr. 50, 349–357 (1997)

27. Zanin, V.: A new nutritional idea for man: lucerne leaf concen-

trate. In: Maj, R., Zioło, Z., Kowalczuk-Vasilev, E. (eds.) Positive

health impact of alfalfa’s leaves extract in human nutrition. Stow.

Rozwoju Regionalnego i Lokalnego ‘‘Progress’’ Dzierdziowka-

Lublin vol. 4, pp. 15–46. (2009)

28. Gafar, M.K., Itodo, A.U., Atiku, F.A., Hassan, A., Peni, I.J.:

Proximate acidic mineral composition of the leaves of Hairy

Indigo. In: 34th Annual International Conference Proceedings of

Chemical Society of Nigeria. 19–23 Sept 2011

29. Nwaogu, L.A., Ujowundu, C.O., Mgbemena, A.L.: Studies on the

nutritional and physico-chemical composition of Amaranthus

hybrids leaves. J. Biores. 4(1), 28–31 (2000)

30. Vitamin A deficiency and Xerophthalmia. Report of a Joint

WHO/USAID Meeting. World Health Organization, Geneva

WHO Tech Rep Ser No. 590 (1976)

31. Kotake-Nara, E., Nagao, A.: Absorption and metabolism of

xanthophylls. Mar. Drugs 9, 1024–1037 (2011)

32. Miller, N.J., Sampson, J., Candeias, L.P., Bramley, P.M., Rice-

Evans, C.A.: Antioxidant activities of carotenes and xantho-

phylls. FEBS Lett. 384, 240–242 (1996)

33. Junghans, A., Sies, H., Stahl, W.: Macular pigments lutein and

zeaxanthin as blue light filters studied in liposomes. Arch. Bio-

chem. Biophys. 391, 160–164 (2001)

Waste Biomass Valor

123

Author's personal copy

34. Yuan, J.P., Peng, J., Yin, K., Wang, J.H.: Potential health-pro-

moting effects of astaxanthin: a high-value carotenoid mostly

from microalgae. Mol. Nutr. Food Res. 55, 150–165 (2011)

35. Sheffner, A.L.: In vitro protein evaluation. In: Albanese, A.A.

(ed.) Newer Methods of Nutritional Biochemistry, with Appli-

cations and Interpretations, Academic Press, New York & Lon-

don (1967)

36. Pirie, N.W.: Leaf Protein and its By-products in Human and

Animal nutrition, 2nd edn. Cambridge University Press, Cam-

bridge (1987)

37. Butler, J.B.: An investigation into some causes of the differences

of protein expressibility from leaf pulps. J. Sci. Food Agric. 33,

528–536 (1982)

38. Subba Rau, B.H., Ramana, K.V., Singh, N.: Studies on nutritive

value of leaf proteins and some factors effecting their quality.

J. Sci. Food Agric. 23, 233–245 (1972)

39. Maliwal, B.P.: In vitro methods to assess the nutritive value of

leaf protein concentrate. J. Agric. Food Chem. 31, 315–319

(1983)

Waste Biomass Valor

123

Author's personal copy