PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF FLOUR OBTAINED FROM THE BULB OF  Lilium brownii

Kain RJ¹, Wang Z² and TS Sonda³

ABSTRACT

 Chemical, physical and functional properties of flour obtained from the bulb of Lilium brownii were studied. Results obtained from analyses using AOAC and phenol-sulphuric acid methods show that lily flour has low protein (13.7%) but high

carbohydrate contents (58.7%), respectively. Blanching had minimal effect on biochemical components investigated. However, there was a significant difference in the colour of flour obtained from bulblets that were blanched and those that were not blanched as the former had a more acceptable colour than the latter. This is indicative of the fact that blanching minimized the occurrence of Maillard reaction. The amino acid composition as determined using Amino Acid Analyzer show that lily flour lacks two of the sulphur amino acids (cystine and cysteine), and contains low lysine (0.77 g/100 g sample) and methionine (0.10 g/100 g sample) contents. Arginine, a basic amino acid that is nutritionally considered as an essential dietary requirement for children but not for adults, is present in a relatively large quantity (2.99 g/100 g sample). Blanching slightly affected the amino acid contents as those samples that were blanched had comparatively lower amino acid contents. Differences in protein composition between defatted sesame flour and lily flour resulted in differences in water and fat absorption capacities as the latter exhibited a lower water and fat absorption capacities than the former. Similarly, whipping properties were influenced by differences in protein contents because sesame flour with higher protein content exhibited superior whipping properties over lily flour.

Analysis of neutral sugar components by Gas Chromatography revealed that lily flour contains predominantly glucose (48.4%) with very low xylose and arabinose contents. Molecular weight distribution was determined using high performance liquid chromatography and results show that lily flour has a molecular weight distribution ranging from 2.91 × 10² to 1.02 × 10⁶. Flour from lily bulb exhibited favourable functional properties such as ability to absorb water and fat, emulsification, foaming and whipping which could be usefully employed in some food preparations.

Key words:Lily, functional properties, Maillard reaction.

INTRODUCTION

 Lily, a common name for a family (liliaceae) comprising more than 250 genera and about 4000 species are mostly cultivated as food crops. These herbs tend to have narrow, parallel-veined leaves and underground storage organs such as rhizomes, bulbs, corns or tubers. The main component of lily bulb is starch. It has a very high starch content but limited in amino acids. Some research findings have shown that lily starch has the same crystalline structure (B-type) as potato starch, but contains an appreciably higher amount of amylase [1].

 Lily has not been widely utilized as food especially in sub-tropical Africa where the need for alternative food sources is of vital importance especially in the 21^st century. However, it has long been popularly used for medicinal purposes. A protein with strong antifungal and mitogenic activities has been isolated from dried bulbs of lily (Lilium brownii). The isolate exhibited an inhibitory action on the activity of HIV-1 reverse transcriptase [2]. As a result of the complexity of its chemical composition, lily is easily affected by Maillard reaction during and after harvesting, during storage and processing into food. Maillard reaction results in browning which may render the colour unacceptable to potential consumers as consumers consider colour as one of the important organoleptic properties.The primary goal of food scientists in this century is to ensure sustainable food supply and availability. It is, therefore, with this view that this work has been undertaken so as to critically analyze the chemical, physical and functional properties of flour produced from the bulb of lily (Lilium brownii) and its application in the food industry.

MATERIALS AND METHODS

 Fresh lily bulbs were obtained from the local market in Wuxi, P. R. China. The lily bulbs were properly washed in distilled water and the fresh bulb-scale segments or bulblets removed. Two methods were used to prepare the lily flour: first, the bulblets were chopped into smaller pieces, dried in a temperature controlled oven for 48 hours at a constant temperature of 45 ºC. The dried-chopped-bulblets were milled into flour using an HR 2839 model Philip blender and the milled flour put into tight containers and stored in a dessicator. In the second method, the bulblets were removed from the bulbs and blanched at 90 ºC for7 minutes using 0.3% citric acid as browning inhibitor. It was removed from the blanching medium and immediately washed with cold water several times to avoid off flavors and to reduce acid concentration. It was then blended into pulp using an HR 2839 model Philip blender and the milled slurry-liquid-like lily was freeze-dried at -20 ºC for 48 hours. The freeze-dried sample was milled using an HR 2839 model Philip blender and stored in a desiccator. The de-hulled sesame seed was purchased from a local market in Wuxi, P. R. China, milled and defatted according to the method used in similar studies [3].The enzyme AS1398 (origin Bacillus Subtilis; type proteinase, cellulase activity > 1x10⁵ μ/g) was obtained from Genecor (Wuxi) Bio-Products Co. Limited, P.R. China. The catalyst 1-methylimidatzole used in the acetylation of monosaccharides and standard dextrans were bought from the Shanghai branch of Sigma Chemical Co. Some of the dextrans obtained from ICN Biochemicals were given to us as a gift.All other chemicals used were of reagent grade (except otherwise stated) and obtained either from other laboratories or from the Chemical Department of Southern Yangtze University, P.R. China.

Fat absorption

 The fat absorption capacity of lily flour was determined using a modified form of the method described by Lin et al. [4]. Samples were weighed into a tared 50 ml centrifuge tube and 30 ml of pure soybean salad oil (in amounts of 5 g produced by East Ocean oils and Grains industries, Zhang Jiagang, Jiangsu Province, P.R. China) added and content thoroughly mixed for 5 minutes, held at room temperature for 1 hour and then centrifuged (2500 x g for 15 minutes). The excess oil was poured off and the tube inverted for 30 minutes. The weight of oil retained was calculated by difference and fat absorption expressed as g oil bound g¯¹ sample x 100%. Estimations were performed in triplicates and the mean value reported.

Water absorption

 Water absorption was measured according to the method described by Sosulski [5]. Estimations were performed in triplicates and mean value reported.

 Bulk density

 The bulk density was determined using the method described by Wang and Kinsella [6]. Estimations were performed in triplicates and mean value reported.

Whipping properties

 The whipping properties of 3% dispersions of lily flour were determined using a modified form of the method described by Lin et al. [4]. Samples (10 g) were dispersed in distilled water (250ml) and pH adjusted to 7.0 using 0.1M NaOH or HCL. The suspension was thereafter homogenized for 1 min at maximum speed level using an HR 2839 model Philips blender and pH checked and adjusted when necessary. Suspensions were then whipped, using maximum speed, in a Kenwood Chef Food Mixer for 10 minutes with the wire whip attachment. The resulting foam was immediately poured into a liter-measuring cylinder and foam height and volume of liquid collecting in the bottom of the cylinder were measured at intervals. The percentage foam expansion was calculated according to the method described by Lawhon et al. [7].Foam volume as percentage was calculated taking the foam volume at zero time as 100%. Leakage was calculated as volume of liquid collected over volume of liquid before whipping x100.

Emulsifying properties

The emulsifying properties and stability were determined according to the methods of Pearce and Kinsella [8] and Matsudomi et al. [9]. For the determination of emulsifying properties, emulsions of oil  and 0.1% of  sample of lily flour and gum Acacia powder (a commonly used emulsifier in foods used as a control) were formed in a ratio of 1:3, respectively. The solutions were shaken and homogenized (24,000 rpm for 1 minute) at room temperature using an ULTRA-TURRAX T25 model homogenizer produced by Janke and Kunkel GmbH and Co. KG, IKA Labortecnik, Staufen, Germany. Aliquots (100 ml) were taken from the bottom of the emulsion after standing for 0, 1, 3, 7, 10, 15, 20, 25 and 30 minutes and diluted with 5ml of 0.1% sodium dodecyl sulphate (SDS) solution. The absorbance of the diluted emulsion was measured at 500 nm. The emulsifying activity was determined from the absorbance measured immediately after emulsion formation (0 minutes). For the determination of emulsion stability each of the emulsions under test were held at constant temperatures of between 60 and 80 ºC in a temperature controlled water bath while being gently stirred. Periodically aliquots of emulsions were taken for dilution and turbidity measurements, as described above.

Chemical methods of analysis

 Crude protein, moisture, crude fiber and ash contents were measured by AOAC methods [10]. Total sugar was determined by the phenol-sulfuric acid method, using glucose as a standard [11]. Using galacturonic acid obtained from the Shanghai Branch of Sigma Chemical Co. as a standard, uronic acid was measured according to the method described by Blumenkrantz and co-worker [12]. To 0.2 ml of sample, 1.2 ml of sulphuric acid/tetraborate was added. The mixture was shaken in a vortex mixer and tubes heated in a water bath at 100 ºC for 5 minutes. After cooling in a water-ice bath, 20 μl of m-hydroxydiphenyl reagent was added. The tubes were shaken and, within 5min, absorbance measurements were taken at 520 nm. The remaining procedures were the same as described by Blumenkratz and co-worker [12], only that 6 ml of urine was used as the specific gravity of the urine was very low. After hydrolysis and conversion ofmonosaccharides produced into alditol acetate derivatives the quantitative neutral sugar component was determined by Gas Chromatography. The sample preparation was carried out according to the method Described by Blakeney et al. [13]. Briefly, the sample preparation was carried out as follows: 10 mg of sample was treated in a test tube with 125 ml of 72% (w/w) H₂SO₄   and dissolution of sample in the acid was accelerated by agitation in a vortex mixer. The test tube was sealed under pressure and placed in a temperature-controlled oven at 121 ºC for 1h.The remaining processes of hydrolysis, reduction of monosaccharides, and acetylation were carried out according to the method used by Blakeney et al. [13]. The pipetted sample, which was stored in 1-ml septrum- capped vials, was used for gas chromatography (GC) analysis. GC was carried out with a shimadzu GC-14A, using the following conditions: OV1701 column (30 x 0.25 mm) with column temperature (195-240 ºC), nitrogen gas carrier (1 ml/min), FID temperature (230 ºC) and injection temperature (250 ºC)

Molecular weight determination

 Seize exclusion-high performance chromatography (SE-HPLC) was performed on a Waters Associates ( Milford, MA 01757) liquid chromatography system equipped with a model 510 pump, WISP model 712 injector and a model 2410 refractive index detector.The detector signal was recorded and integrated by a Data module integrator, waters 740. An ultrahydrogel guard column followed size-exclusion ultrahydrogel linear (7.8 mm i.d. x 300 mm) and 2 ultrahydrogel 120 columns (120 Å in pore size). Elution was with 0.1N NaNO₃ at a flow rate of 0.4 ml min¯¹at 40 ºC. The sample was solubilized in 0.1N NaNO₃   solution at 35 ºC for 30 minutes. After solubilization, the sample under test was centrifuged (1500xg for 10 minutes) and filtered through a 0.45 mm filter under vacuum. The clear supernatant solution (20 ml) was injected into the SE-HPLC column. The calibration equation was obtained by linear regression of a plot of retention time against log molecular weight of standard. The standard plot was obtained by injecting 5 dextran standards with varying molecular weights (1x10⁴ sigma company; 7.2x10⁴ 1CN Bio-chemicals; 2x10⁵ 1CN Bio-chemicals; 5x10⁵ Sigma company; 2x10⁴ Sigma company) into the SE-HPLC unit.

Amino acid analysis

 Lily flour was hydrolyzed for 24 hours by refluxing in 6 N HCl, evaporated to dryness, and dissolved in citrate buffer at pH 2.2. A portion of the hydrolyzate with norleucine as internal standard was assayed in H835-50 HITACHI Amino Acid Analyzer. 

Enzymatic hydrolysis

 Lily flour was defatted at 40 ºC with 5 times the volume of n-hexane. The extraction of protein from sample was carried out by enzymatic hydrolysis. The protein was hydrolyzed twice with AS1398 Enzyme. Five times the weight of distilled water was added to the sample and homogenized. The enzyme was added to the homogenate in a bio-reactor at 1.0% of the weight of the solid sample. The suspension was maintained at constant temperature and pH of 38 ºC and 7.0, respectively, for 4 hours while continuously stirring for the protein to be hydrolyzed. The hydrolyzed and dissolved proteins were removed by centrifugation (5,000 x g for 30 minutes). The resulting precipitates were freeze-dried and used for the analysis of neutral sugar components and molecular weight distribution.

RESULTS

Effect of pretreatment methods on the colour of lily flour

 The colour difference between the flour obtained from the twotreatment methods was distinct indicating that bulblets that were blanched underwent minimal Maillard reaction, hence the colour of flour obtained was off-white, while that for bulblets that were not blanched  was brownish.

Biochemical scores

 Results in Table 1 show that the pretreatment methods had minimal effect on biochemical scores obtained. Results also show a concentration of biochemical components measured in treated samples as compared to those obtained from the fresh samples. While the concentrations of the intrinsic nutritional components, such as protein, fat, carbohydrate, ash and crude fiber increased in the treated samples as compared to fresh samples, those for moisture content were decreased. Fresh samples had a moisture content of 56.0% while those for oven dried and blanched, freeze-dried samples were 15.5% and 15.7%, respectively. However, carbohydrate forms a major intrinsic component of lily flour (≈59%).

Bulk density, fat and water absorption

Data on the above functional parameters are shown in Table 2. Oven dried lily flour had higher bulk density (0.384 g ml^-1) than defatted sesame flour (0.233 g ml^-1), while the former had a lower bulk density than flour obtained from the blanched sample (0.432 g ml^-1). Fat and water absorption capacities were higher in defatted sesame flour than in lily flour obtained from the two processing methods.

Whipping properties

Whipping properties compared in Table 3 show that defatted sesame flour gave the highest foam expansion (480%)  and stability and the highest initial foam volume (130%).  It should be noted that both foam volume and foam leakage were measured as indices of foam stability. Increase in foam leakage, as could be observed in Table 3, was not always accompanied by corresponding decrease in foam volume as a result of adherence of foam to the sides of the vessel. During the experimental period the blanched and freeze-dried lily flour exhibited the highest stability compared to the other two samples studied. However, defatted sesame flour showed the highest stability during the initial observation stage.

Emulsifying properties

 Emulsifying properties of lily flour, defatted sesame flour and gum acacia (used as control) are shown in Figure 1. Defatted sesame flour exhibited high emulsifying properties at the initial experimental stage. Defatted sesame flour and gum acacia had almost the same emulsifying properties over the experimental period. Lily flour exhibited a lower emulsifying property although it was slightly more stable over experimental time.

After 30 minutes the samples under discussion had almost the same emulsifying property.

  Figure1: Emulsifying properties of lily flour, defatted sesame flour and control (gum acacia powder)

Heat stabilities of lily flour, defatted sesame flour and gum acacia at 60 and 80 ºC are shown in Figure 2. Emulsions were stabilized by 0.1% SDS solutions and heated  at 60 and 80 ºC. All samples exhibited high emulsifying properties at 60 ºC. However, the samples showed complete instability over experimental time. Sesame and gum acacia had the highest emulsifying properties at the initial stage but were completely unstable over the experimental period. At 80 ºC all samples exhibited low emulsifying properties.

  Figure 2: Heat stability of lily flour, defatted sesame flour and control (gum acacia powder). Emulsions stabilized by 0.1% SDS solutions heated  at 60 and 80 ºC

Amino acid analysis

 Amino acid components of samples obtained from different treatment methods are shown in Table 4. Two of the sulphur amino acids, cystine and cysteine, are not found in lily flour. Methionine (0.10 g/100 g sample), phenylalaline (0.45 g/100 g sample), isoleucine (0.31 g/100 g sample), leucine (0.77 g/100 g sample) and lysine (0.71 g/100 g sample) are found in small quantities in lily flour. Arginine (2.99 g/100 g sample), aspartic acid (1.07 g/100 g sample) and glutamic acid (0.99 g/100 g sample) are found in relatively high quantities. Results also show that pre-treatment methods had an effect on amino acid components of samples as comparatively higher components were found in oven dried lily flour.

Neutral sugar composition of lily flour

 Neutral sugar composition of lily flour is presented in Table 5. Glucose (48.4%) and galactose (22.1%) were found in large quantities in lily flour. Other neutral sugar components such as mannose (5.3%), xylose (2.3%) and arabinose (1.7%) were found in minute quantities. Results also show that the lily flour contains 3.8% protein and an ash content of 4.8% after enzymatic hydrolysis.

Molecular weight distribution

 Molecular weight distribution of lily flour is given in Table 6. The molecular weight was calculated according to the standard equation below:

 LogM_w = - 0.439_RT + 13.013                r = 0.9986

 here M_w = molecular weight and R_T = retention time (min)

 Peaks one and two had the highest molecular weights of 1.02x10⁶ and 7.07x10⁵ Da, respectively.

DISCUSSION 

Effect of pretreatment methods on the colour of lily flour: One of the major problems affecting the use of lily bulb as food is the rapid rate at which maillard reaction occurs. The optimum pre-treatment conditions established yielded encouraging results with respect to the colour of the flour produced from lily bulblets that were blanched by heating at 90 ºС for 7 minutes in 0.3% citric acid as blanching agent. The possibility for the occurrence of browning was tremendously reduced thereby resulting in a favourable colour – an important organoleptic property for consumers to accept a food product. The reducing sugar-protein reaction is referred to as Maillard reaction or non-enzymatic browning. Blanching partially denatures proteins which consequently minimizes the reducing-sugar protein reaction, hence resulting in the occurrence of minimal browning.

Biochemical scores

 The two treatment methods (oven drying and freeze drying after blanching) have comparable effect on the biochemical composition of flour made from lily bulbs. After processing fresh lily bulb into flour, the chemical components became concentrated, hence the high percentage composition in the former as compared to the latter. The flour contains favourable nutritional components such as protein, fat, carbohydrate, ash and crude fiber, with carbohydrate forming a principal component which is in accordance with earlier findings [1].

Bulk density, fat and water absorption

 Differences in particle size, as could be seen in Table 2, may have contributed to differences in bulk densities. The defatted sesame flour had a lower bulk density than lily flour which can be attributed to the fact that the former had larger particle size than the latter. A similar relationship between particle size and bulk density has been reported [14]. Differences observed in both fat and water absorption capacities of samples studied may have been due to both protein composition ie number of exposed hydrophilic and lipophilic groups, and ability to physically trap liquid as reflected in the bulk densities. Fat acts as a carrier for organic molecules that give food products their characteristic flavour and aroma and markedly contribute to their texture, that is, plasticity and smoothness of solid or semi-solid fats, the creaminess, and oily mouth feel of food emulsions. Large consumption of fats coupled with lack of physical activities is considered responsible for obesity, which induce other diseases such as artherosclerosis, hypertension, diabetes and some forms of cancer [15]. Hence demand for food with low fat content (low fat foods) has increased over the years. Since lily flour has a comparatively lower ability to absorb fat, it could be usefully employed in food preparations where low fat contents are required. Besides, lily flour also has acomparatively lower ability to absorb water thereby rendering it useful in preparing food items that require low moisture. This reduces water activity (a_w) which consequently reduces microbial activity thereby increasing the shelf life of the food. Such applications may be very useful in the preparation of confectionaries.

Whipping properties

 It should be noted that differences in protein contents of samples contributed tremendously to differences in whipping properties. Defatted sesame flour, with high protein content, exhibited superior whipping properties over lily flour with comparatively low protein content. Similar relationship between protein content and whipping properties of samples has been reported [7].

Emulsifying properties

 Emulsifying properties are normally attributed to the flexibility of solutes (such as the ability to go into solution and adsorb to interfaces, and the exposure of the hydrophobic domains). This means that the hydrophobic hydrophilic balance becomes an important parameter in determining emulsifying property and stability of solutes. Sesame flour is less soluble which increases its tendency to have high affinity for both oil and water thereby enhancing its emulsifying property and stability (Figure1). Besides, the content of protein-polysaccharide conjugate in sesame flour is higher than in lily flour, which may have also served as a major contributing factor to the higher emulsifying property and stability exhibited by the former (Figure 2). In any case sesame flour and gum acacia (a commonly used emulsifier in foods) used as control had only a slightly higher emulsifying property and stability than lily flour. This is indicative of the fact that, to some extent lily flour could serve as a potential alternative source of emulsifier for different food preparations such as confectionery products.

Amino acid analysis

 Lily lacks two of the sulphur amino acids (cystine and cysteine) that are not actually regarded as essential because they can be made from methionine which is present but in a relatively low quantity, about 0.11 g/100 g protein (Table 4). Similarly the lysine content and other essential amino acids such as phenylalaline, isoleucine, leucine and valine are limited in lily flour, which is in agreement with similar studies [1]. However, arginine, a basic amino acid that is not a dietary essential for adults, although essential to a certain extent in the promotion of growth in children, is present in a fairly large quantity in lily. Also the treatment method seemed to have had some effect on the amino acid profile in Table 4. Amino acids obtained as a result of blanching were comparatively lower than those obtained from just chopping and drying of lily bublets. 

Neutral sugar composition of lily flour

 The hydrolysis process was effective in the denaturation of lily protein as shown in Table 5. The dominant neutral sugars in lily are glucose and galactose while the other neutral sugar components mannose, xylose and arabinose are present in minute quantities. The high glucose content of lily flour has been reported in similar studies [1] which is indicative of the fact that lily flour can serve as a potential energy source especially in third world countries where there is an acute shortage of energy food sources.

Molecular weight distribution

 A vast difference was found in molecular weight distribution between peaks  (1.02x10⁶ Da) and four (2.91x10² Da). Further studies are recommended to evaluate the nutritional importance of  molecular weight distribution of polysaccharides in lily flour.

CONCLUSION

 Results obtained show that lily contains some intrinsic and extrinsic food properties. However, most of the bulk comprises starch - glucose. Lily bulb is affected by Maillard reaction, therefore some careful blanching is required if it has to be used as a food. Lily flour has a low capacity to absorb fat and water and is therefore suitable for application in food preparations that require low fat and/or water content. Lily flour exhibited a slightly lower emulsifying property and stability than gum acacia. However, lily flour can serve as a relatively cheap source of emulsifier for different food preparations such as confectioneries. The results also show that lily flour has a molecular weight of approximately 1.02 x 10⁶. Further research on the nutritional implication of the amino acid profile and neutral sugar composition, through animal bio-assay is strongly recommended.

Table1: Biochemical scores of lily samples

                                                   Biochemical scores (%)

Fresh lily bublets                            4.3        56.0          0.2          35.2         1.2         3.1         

Oven dried sample                        13.7        15.5         1.2           58.9        3.8         6.9

Blanched, freeze-dried sample     13.9        15.7          1.3          58.7         3.6         6.8

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Table 2: Comparative analysis of some functional properties of oven-dried, blanched and freeze-dried samples and defatted sesame flour. All results obtained in triplicate and mean values recorded.

 Table 3: Whipping properties of different pretreated lily flour and sesame flour

 1 = oven dried lily flour; 2 = blanched and freeze-dried lily flour; 3 = defatted sesame flour

   Table 4: amino acid composition of lily flour                                   

Table 5: Neutral sugar composition of lily flour

_______________________________

Protein (%)                 3.84

Ash      (%)                 4.79

                                                  Sugar (%)                     -

Glucose                      48.4

Galactose                    22.1

Mannose                     05.3

Xylose                        02.3

Arabinose                   01.7

Uronic acid                 15.2

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Table 6: Molecular weight distribution of lily flour

Peak number                       Molecular weight (Da)

________________________________________

1                                           1.02x10⁶

2                                           7.07x10⁵

3                                           8.64x10²

4                                           2.91x10²

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¹Foreign Students' & Experts' Building – 413, Southern Yangtze University, Wuxi – 214036, P. R. China. Tel: +86-510-587664 or +86-13921543381, Fax: +86-510-5876646. E-mail: regenakain@hotmail.com or regenakain@sierraleonelive.com

²School of Food Science & Technology, Department of Food Science, Southern Yangtze University, Wuxi – 214036, P. R. China. Tel: +86-510-588449 Fax: +86-510-5807976. E-mail: syxu@wuxi.edu.cn

³c/o Regena J. Kain, Foreign Students' & Experts' Building – 413, Southern Yangtze University, Wuxi – 214036, P. R. China. Tel: +86-510-5876646, Fax: +86-510-5876646. E-mail: tambasonda@hotmail.com