J. Agrlc. Food Chem. 1085, 33, 79-83
79
Functional Properties of Fraction 1 Protein from Tobacco Leaf Shuh J. Sheen* and Vera L. Sheen
Crystalline fraction 1protein (F-1-p) from tobacco leaf was spray-dried in a crystal suspension and in a pH 8.5 solution or freeze-dried after solubilization at pH 3. The nitrogen solubility was higher for all three preparations than for soy protein isolate. The latter was also inferior to the pH 8.5 and pH 3 F-1-p in water and fat absorption. Emulsions in the form of pudding and mayonnaise with the pH 3 F-1-p were stable. Sodium chloride improved the emulsification of the crystal and pH 8.5 F-1-p but not the pH 3 preparation. The latter ranked the highest in foaming capacity and stability. F-1-p and egg white possessed a similar whipping property. Upon baking, whipped cream of the leaf protein resembled egg meringue. The pH 3 and pH 8.5 F-1-p in 3% solution with and/or without NaCl gelled instantaneously at near-boiling temperature. F-1-p from tobacco leaf could, therefore, be a valuable protein source in food formulations in view of its highly desirable functional properties.
Pirie (1942) promoted the idea of using leaf proteins for human food and developed a simple technique for isolating them from various plant species. The protein so isolated was a green coagulate. Recently, Knuckles and Kohler (1981) developed a process to separate green aggregates from the edible “white” protein fraction in alfalfa leaf extract. This white leaf protein not only contains a balanced composition of amino acids but also possesses certain desirable functional properties that make it suitable in formulated food systems (Knuckles and Kohler, 1982). However, the edible white protein from alfalfa contains only 88.6% crude protein. In green leaves of many plant species, more than 50% of the soluble protein is fraction 1protein (F-1-p), which accumulates inside chloroplasts (Kawashima and Wildman, 1970). The term F-1-p was coined by Wildman and Bonner (1947), and this protein was later identified as ribulose-l,5-bisphosphatecarboxylase-oxygenase, which catalyzes COPfixation (Weissbach et al., 1954) and photorespiration (Marsho and King, 1976). Leafy vegetables, containing F-1-p, offer a good protein for human nutrition. The steady increase in world population has necessitated a search for new sources of protein to meet growing demands. The naturally abundant substance F-l-p deserves attention from researchers, manufacturers, and consumers as well. A new technology has just been developed by Leaf Proteins, Inc., of California to isolate crystalline F-1-p from young tobacco plants at a pilot-plant scale (Wildman, 1983). The protein crystals are soluble in water, tasteless and odorless, and composed entirely of amino acids. When fed to rats, tobacco F-1-p consistently exhibited a higher nutritional performance than casein, the standard commonly used for comparing the nutritional quality of proteins (Ershoff et al., 1978). The potential of growing tobacco as a food crop rests in part on (i) the yield and value of edible protein, mainly F-1-p, from tobacco biomass produced on cultivated land, (ii) the quality of F-1-p in terms of functionality for suitability in food formulations, (iii) the economic value of biomass residues that can be used as animal feeds (Sheen, 1983) or processed to a safer smoking material (Wildman and Sheen, 1981),and (iv) the cost of biomass production. For leaf protein production, tobacco will be grown under high density and harvested as “ratooning” crops repeatedly during the growing season. This agricultural practice eliminates more than 50% of the Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546. 0021-656118511433-0079$01.50/0
labor requirements for the conventional production of tobacco leaf. With high-density growth and multiple harvest, it is possible to yield 600 kg of F-1-p ha-’ year-’ in the U.S. tobacco-growing regions (Lowe and Sheen, 1982). This paper reports our investigation in the functional properties of tobacco F-1-p. The effects of the methods of protein preparation prior to dehydration on functionality were evaluated, and the results were compared with those of soy protein isolates and egg white. The effectiveness of nicotine removal from the leaf protein by the washing process was also measured. MATERIALS AND METHODS Source and General Property of Proteins. Bright tobacco (Nicotiana tabacum L.) cultivar NC 95 was grown under high density (approximately 500 OOO plants/ha) by direct seeding in North Carolina in 1982. When the plants reached 60 cm in height, the biomass was harvested by a green chopper and processed to obtain crystalline F-1-p in the pilot plant of Leaf Protein International, Inc., of North Carolina (Wildman, 1983). The protein, either in crystal suspension or in pH 8.5 solution, was dehydrated by a spray-dryer equipped with a rotary atomizer and cyclone collector. The inlet and outlet temperatures were 220 and 85 “C, respectively. The same protein preparation was also freeze-dried after solubilization at pH 8.5 with NaOH and then shifting to pH 3 with HC1. Soy protein isolates and egg white were compared with the tobacco F-1-p preparations by using various functional property tests. The soy protein isolates, namely, Ardex R (pH 4.5) and Ardex F (pH 7) (Archer Daniels Midland Co.) and All Star (Natural Sales Co.), were obtained as a gift or purchased from the supermarket; the egg white was ordered from Sigma Chemical Co. The nitrogen percentage in the proteins was analyzed with micro-Kjeldahl digestion followed by Berthelot’s reaction to quantitate ammonia using ammonium sulfate as the standard (Bradstreet, 1965). Moisture content was measured by the standard method of oven-drying the proteins at 102 OC for 3 h and weighing the weight loss after cooling in a desiccator. Functional Properties. Analyses of solubility, water and fat absorption, emulsification, foaming, whipping, and gelation or heat-setting were performed in triplicate for each protein sample, unless otherwise stated. The solubility as a function of pH was determined by Betschart’s method (1974) with the modification that the protein quantity in the supernatant was assayed with Bio-Rad protein reagent (Bradford, 1976) by using bovine serum 0 1985 American Chemical Society
80
Sheen and Sheen
J. Agric. Food Chem., Vol. 33, No. 1, 1985
Table I. Composition of Tobacco Fraction 1 Protein, Egg White, and Soy Protein Isolates” crude moisture, nitrogen, protein,* protein % 9i % ash, % crystal F-l-p 5.3 15.2 95.0 3.5 pH 8.5 F-l-p 6.1 15.1 94.1 4.2 pH 3.0 F-l-p 6.0 14.7 92.3 6.0 egg white 4.6 14.6 91.1 4.3 soyprotein (All Star) 4.7 14.5 90.5 4.0 soy protein isolate 6.0 14.6 91.0 4.3 (Ardex F) soy protein isolate 6.2 14.7 92.0 3.9 (Ardex R)
40
-
20
-
bp
v
4
,..n
“Except for moisture, data are reported on a dry weight basis.
b N X 6.25.
albumin as a standard. The effect of NaCl concentration on protein solubility was also studied with the pH 8.5 and pH 3 protein preparations at a range of pH 2 to pH 11. Water and fat absorption capacity was measured by the methods of Fleming et al. (1974) and Lin and Humbert (1974),respectively. The weight gain of the protein powder presoaked in 0.1 M phosphate buffer, pH 5, or corn oil signifies the amount of water or fat absorbed. Lawhon and Carter’s (1971) method was used to determine the foaming capacity and stability that were recorded at given time intervals. The emulsifying property of the proteins was determined by a procedure similar to that described by Yamauchi et al. (1980). A mixture of 1%, 2%, 4%, and 6% protein in 100 mL of distilled water containing 20% or 40% corn oil was homogenized in a VerTis homogenizer at a speed of 20000 rpm for 8 min. The viscosity of the emulsion expressed in centipoises was measured with a Stormer viscometer at 25 OC, with glycerin as the standard. The effect of the addition of NaCl and starch on viscosity was also investigated. The whipping property was determined by whipping 3 g of protein in 100 mL of distilled water at high speed for 6 min. The resultant volume of whipped cream was recorded according to Lawhon and Carter’s method (1971). The cream was whipped again for an additional 2 min after adding 75 g of sugar, and its final volume was recorded. The percent increase in volume was calculated from the amount of volume increase after whipping and whipping with added sugar to the initial volume prior to whipping. The sweetened whipped cream was put on lemon pie fillings and baked in an oven for 5 min at 425 OF. The resultant meringue pie was compared with that made from egg white in a similar manner. In the determination of gelation and heat-setting properties, suspensions of various protein concentrations were placed in a water bath and gradually heated to a boil. The temperatures at which the protein instantaneously gelled as a heat-setting phenomenon were recorded. Since a penetrometer was not available for the measurement of gel strength, the relative gel rigidity was scored with reference to a concentration series of gelatin gels. This was determined by the penetration rate of a glass rod into the gel due to gravity. RESULTS
General Chemical and Physical Properties. F-l-p from tobacco leaf had a white to off-white appearance, irrespective of spray-drying or freeze-drying. The bulk density of spray-dried F-l-p in the range of 0.4-0.6 g/cm3 was about 5 times greater than the freeze-dried F-l-p and comparable to that of soy protein isolates and egg white. Among the proteins analyzed, crystal F-l-p had the highest concentration of crude protein, followed by that of pH 8.5
5
“7
+ 0 &
s o 50-
0
soy i s o l a t e , PH 4 . 5 Soy isolate, pH 7.0
2
3
4
5
6
7
a
PH
Figure 1. Solubilityof tobacco fraction 1protein and soy protein isolates as a function of pH.
and pH 3 (Table I). The crude protein content in soy protein isolates and egg white was 92% or less by weight. With the exception of pH 3 F-l-p, the ash content was comparable among the various proteins. The ash content of three F-l-p preparations appeared to be inversely related to the crude protein concentration. This reflects the effects of salts, mainly sodium metabisulfite, that had been added to the crystal suspension in a 0.1 % concentration prior to its shipping to other locations for drying. Other contributors to ash content are the inorganic acid and base used for protein solubilization. To ascertain such possibilities, samples of all three F-l-p preparations were solubilized at the appropriate pH and dialyzed in several changes of distilled water. The retentates were freeze-dried and quantitated for percent nitrogen by the same procedure mentioned earlier (Bradstreet, 1965). The average crude protein concentration of the dialyzed samples was 99.3%. It is therefore certain that commercial production of tobacco F-l-p by spray-drying at the production site could obtain a protein purity of 99% or higher since the use of sodium metabisulfite becomes unnecessary. Solubility Property. Protein solubility profiles as a function of pH are given in Figures 1 and 2. Except at pH 6, tobacco F-l-p could be solubilized in a greater quantity than soy protein isolates. pH 3 F-l-p was completely soluble at pH 3, whereas the other two preparations were nearly 90% soluble at pH 8. Protein solutions dehydrated by spray-drying or freeze-drying showed no difference in solubility. In the presence of NaC1, the solubility of both pH 8.5 and pH 3 F-l-p decreased as the salt concentration increased. Nevertheless, their solubilities in the edible range of food salinity remain higher than for soy protein isolates in most pH ranges. Water and Fat Absorption and Emulsification. In order to minimize the effect of bulk density on the water and fat absorption, all samples were sieved through a 60-
J. Agric.
Properties of Fraction 1 Protein from Tobacco Leaf
Table 11. Water and Fat Absorption of Tobacco Fraction 1 Protein water fat protein absorption, % absorption % crystal F-1-p 180.7 105.8 pH 8.5 F-1-p 319.0 304.5 pH 3.0 F-1-p 393.3 375.5 soy protein (All Star) 272.1 187.5 soy protein isolate (Ardex F) 276.3 244.6 soy protein isolate (Ardex R) 191.8 143.4
Food Chem., Vol. 33, No. 1, 1985 81
1 O@
~
Table 111. Emulsifying Property of Tobacco Fraction 1 Protein" viscosity, cP,for protein concentration protein and emulsifying condition 1% 2% 4% 6% 20% oil only crystal F-1-p 25 8 12 8 pH 8.5 F-1-p 6 22 45 99 pH 3.0 F-1-p 43 281 539 5941 442 696 soy protein (All Star) 35 127 20% oil + 1% each of NaCl and starch 8 28 28 55 crystal F-1-p pH 8.5 F-1-p 28 55 133 159 9 pH 3.0 F-1-p 4 11 139 soy protein (All Star) 5 10 29 55 40% oil only 45 97 crystal F-1-p 18 26 pH 8.5 F-1-p 117 284 30 33 pH 3.0 F-1-p 44 535 10379 >50000 soy protein (All Star) 40 140 978 18915 40% oil + 1% each of NaCl and starch 21 41 crystal F-1-p 166 690 pH 8.5 F-1-p 55 126 343 681 26 30 112 146 pH 3.0 F-1-p soy proteidn (All Star) 30 52 151 481
" Average of duplicate analyses. mesh screen before testing. The absorption capacity expressed as an increase in percent weight is presented in Table 11. The protein powder of the pH 8.5 and pH 3 preparations absorbed water and fat at a higher capacity than soy protein isolates. In contrast, crystal F-1-p was poor in these properties. The results of the emulsifying property were consistent with those of fat absorption capacity (Table 111). In all cases, the apparent viscosity of emulsion was increased with the increase in protein and oil concentrations. At least one preparation of tobacco F-1-p emulsified better
e
