Composition and fractionation of sunflower meal: Use of the lignocellulosic fraction as substrate in solid-state fermentation

$Page 1: Composition and fractionation of sunflower meal: Use of the lignocellulosic fraction as substrate in solid-state fermentation$
Biological Wastes 32 (1990) 225-233

Composition and Fraetionation of Sunflower Meal: Use of the Lignocellulosic Fraction as Substrate in Solid-State

Fermentation

Juan Bautista , Juan P a r r a d o & Alber to M a c h a d o

Departamento de Bioquimica, Bromatologia y Toxicologia de la Universidad de Sevilla, 41012 Sevilla, Spain

(Received 24 July 1989; revised version received 9 September 1989; accepted 14 September 1989)

ABSTRACT

The chemical composition of undehulled sunflower meal ( SFM ) was studied in order to evaluate its biotechnological potential as a fermentation source. SFM can be fractionated into three main components, a lignocellulosic fraction ( LCF), a proteinaceous fraction ( PF) and a soluble fraction ( SF), which represent 23.2-25.3%, 55"4-57"6% and 17.1-21.4% of the dry weight, respectively. After removal of the PF, the remaining subproducts ( LCF and SF), have a high potential for use as fermentation sources. Sunflower meal- LCFis a suitable fermentation source for solid-state fermentation ( SSF), as is shown by the growth of different fungi. Trichoderma harzianum S/G2413 growing for 96 h in a sunflower meal-LCF medium produced a protein enrichment of approximately 20%.

I N T R O D U C T I O N

In Spain, large quantities of undehulled sunflower meal (SFM) (c. 700000 t year- 1) are generated from the industrial processing of sunflower seeds into edible oils, and this produces the largest portion of the local organic agricultural byproducts (Anon., 1988). SFM is a part of the whole sunflower seeds which remains after the extraction of the oil by mechanical- or solvent- extraction processes. This byproduct can be fractionated into a proteinaceous fraction (PF), a lignocellulosic fraction (LCF), and a soluble fraction (SF) (Kinard, 1981).

225 Biological Wastes 0269-7483/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain

226 Juan Bautista, Juan Parrado, Alberto Machado

The actual use of SFM is as a supplement in animal feed. However, its high lignocellulosic materials content limits the use to ruminants and poultry. The efficient separation of the PF from the rest would result in a more efficient use of this by-product. The PF could be used for animal feed, as a rich protein source, and the other components, LCF and SF, due to their chemical composition, could be used as potential fermentation sources. This will be discussed in this paper.

METHODS

Analytical procedures

Samples were dried in a vacuum dryer at 60°C for 24 h, and ground through a 100/~m sieve prior to analysis. Total and mineral nitrogen content was determined by the Kjeldahl procedure. The crude protein content value was calculated by subtracting mineral nitrogen from total nitrogen and multiplying the results by 5"5 for the SFM and LCF (Gassmann, 1983) and 6.25 for the enriched LCF (Parrado et al., 1990). Dry matter, pH, crude fat, ash, calcium and phosphorous concentrations were determined according to standard AOAC methods (AOAC, 1980). Neutral detergent fibre (NDF), acid detergent fibre (ADF), sulphuric acid lignin, and acid detergent insoluble nitrogen were determined according to Goering & van Soest (1973) and Cherney et al. (1985), except that sodium sulphite was omitted from the neutral detergent fibre solution, and preheated fibre-glass--instead of asbestos--was added before treatment with 12M H2SO 4. Hemicellulose concentration was estimated by calculating the difference between N D F and ADF, and cellulose concentration was estimated by subtracting the acid- insoluble ash value from the residue remaining after lignin extraction. Non- structural carbohydrates were extracted with deionized water (soluble carbohydrates), or were hydrolyzed using amyloglucosidase (insoluble carbohydrates) (Smith, 1981). Non-structural carbohydrates are expressed on a glucose-equivalent basis.

The N D F was hydrolyzed using a mixture of cellulolytic and hemicellulolytic enzymes as described by Heredia-Moreno et al. (1987), and the carbohydrate composition was determined in a Perkin-Elmer gas chromatograph (model 3720) fitted with a Perkin-Elmer 651 recorder and a Perkin-Elmer M-2 integrator. A flame ionization detector and a 2 m x 2.5 mm ID stainless column packed with Chromosorb W, 80-100 mesh was used. The stationary phase was ECNSS-M (3%). Nitrogen was used as carrier gas. The injector temperature rose from 175°C to 200°C, at a heating rate of I°C min- 1. The internal standard method was used to calculate the percentages of each sugar.

Sunflower meal as a fermentation substrate 227

Microorganisms

Trichoderma harzianum S/G2413, a mutant obtained from Trichoderma harzianum (Coleccion espafiola de cultivos tipo, Universidad de Valencia, Burjasot, Valencia, Spain) was used in the fermentations. This strain had been selected on the basis of certain criteria: protein content, amino acid composition, low nucleic acids content, no toxicity, no antibiotic production, and ability to consume different substrates (Parrado et al., 1989). Cultures were maintained on potato dextrose agar and stored at low temperature.

Inoculum preparation

Dry sunflower meal LCF was moistened with SF/Czapek mineral solution in the proportion 1:3.5 (w/w) and the pH was adjusted to 5.0 with sulphuric acid. Next, 20g of moist LCF + 0.5 g KH2PO4 was placed in a 250ml Erlenmeyer flask and stored for 12 h at low temperature to equilibrate the humidity of the substrate. The flasks were then sterilized at 121°C for 30 min, after which the medium was seeded with 5 ml spore suspension, obtained as described by Duran & Chereau (1987). The cultures were incubated for 3 days at 28°C with vigorous agitation, and used as seed material for SSF.

Media for solid-state fermentation

The constituents of the media for SSF are shown in Table 1. Dry sunflower meal LCF (100 g) was moistened with SF/Czapek mineral solution in the proportion 1:3"5 (w/w), supplemented with urea or ammonium sulphate and potassium phosphate, and the pH was adjusted to 5.0 with sulphuric acid. The moist LCF was placed in a 2000 ml Erlenmeyer flask and stored for 12 h at low temperature to equilibrate the humidity with the substrate. The flasks were then sterilized at 121°C for 30 min, then cooled and inoculated with the 3-day-old inoculum culture and grown with vigorous agitation at 28°C for 96h.

T A B L E 1 Fe rmen ta t ion Media for the G r o w t h of Trichoderma harzianum S/G2413

Medium LCF Urea (NH)4SO 4 KH2 P04 SF/Czapek ~ mineral solution

L C F 100 b - - - - - - 350ml L C F / U - P 100g 3"0g - - 3"5g 350ml L C F / N H 4 - P 100g - - 5"0g 3"5g 350ml

9 volumes of SF and 1 volume of Czapek mineral solution. b Dry weight.


RESULTS AND DISCUSSION

Chemical characterization of sunflower meal

The characteristics of sunflower meals are largely determined by the oil extraction process from which they are derived. Variations occur due to differences in seed, amount of hulls removed in processing, and different processing methods, such as mechanical- or solvent-extraction. As a result, sunflower meal may contain 28-40% crude protein and 15-25 % crude fiber (Angelova & Dimitrova, 1977; Dimitrova, 1977; Gassmann, 1983).

The composition of andalucian industrial sunflower meal is depicted in Table 2. This table illustrates the chemical composition and the differences in meals obtained by mechanical and extraction processes, The analyses are representative only and are based on four different composite samples of four different five-day production runs from each of two sunflower oil mills. The fractional composition of the fiber is also presented in Table 2. The main component in both classes of SFM is lignin, which represents approximately 11-12% of the dry matter.

TABLE 2 C o m p o s i t i o n o f S u n f l o w e r M e a l a f r o m T w o Dif fe ren t P roce s se s

Sunflower meal Sunflower meal mechanically extracted solvent extracted

(%) (%)

D r y m a t t e r 94.70 ___ 0.56 92.27 ___ 0-45

M o i s t u r e 5.30 + 0.56 7"73 + 0.45

A s h 6"80 + 0.10 7.47 ___ 0.15

C r u d e p r o t e i n (N x 5"5) 23"33 + 0 '38 25"57 ___ 0-57

F a t 12.63 _ 0-31 4.00 + 0"82 N e u t r a l d e t e r g e n t fibre 23-70 ___ 0 '56 25"47 ___ 0"31

L ign in 11.00 + 0 '50 11"97 + 0"30

Ce l lu lose 6.52 + 0"20 6"80 + 0-20

Hem±cel lu lose 4"15 ___ 0.25 4.00 ___ 0"30

O t h e r s u b s t a n c e s 2.03 __+ 0-25 2.70 + 0.25

N F E b 26"47 _ 1.10 32'17 ___ 0 '73 So lub le s u g a r s c 3"70 _ 0-20 4"35 ___ 0"25

N o n - s o l u b l e s u g a r s c 13.35 + 0"50 14-05 -I- 0.40

O t h e r s u b s t a n c e s 9"42 ___ 0"35 13.77 + 0"30

P h o s p h o r u s 1"45 ± 0"25 1"55 ___ 0-30

C a l c i u m 0"85 + 0"12 0"96 ___ 0"15

a U n d e h u l l e d sunf lower .

N i t r o g e n - f r e e ext rac t .

c G l u c o s e e q u i v a l e n t bas is , see M e t h o d s .


Solvent-processed sunflower meal is the main byproduct in Andalucia. For that reason we selected this byproduct as a possible fermentation substrate.

In Table 3, the essential amino acid composition of solvent-extracted sunflower meal is compared with that of hens' eggs and with FAO-reference protein (FAO, 1970; Gassmann, 1983). Lysine is the only limiting amino acid in solvent-extracted meal. This deficiency could limit the use for human food and animal feed, but this limitation does not represent any problem for the growth of microorganisms, since the lysine is only slightly lower than that of casein (FAO, 1970a, 1970b; Cheftel, 1989), and casein is a good nitrogen source for many microorganisms (Zabriskie et al., 1982).

So if we efficiently separate the PF of SFM, we have a rich protein source (50-60%) that can be easily and considerably improved by admixing lysine or proteins of high lysine contentfor use in animal feed or used as a nitrogen source in fermentation processes (Bautista et al., unpublished results). This protein source can be used as raw material, PF, or after a hydrolytic process to improve its solubility and other properties (Parrado et al., 1990).

Fractionation of sunflower meal

SFM was fractionated by a sedimentation/flotation process into three fractions, PF, SF and LCF. The PF was sedimented at the bottom of the tank, while the LCF floated at the top. The process must be run as rapidly as possible to reduce the sedimentation of some of the LCF by wetting. Once the LCF was mechanically removed, the SF and PF were separated by filtration.

The results obtained are shown in Table 4. The protein fraction represents more than the 50% of the initial product, while LCF represents 23.2-25.3 %, and the SF 17.1-19.3%.

Chemical characterization of sunflower meal LCF

Sunflower meal LCF, like other collectable plant resources, may be considered for the production of chemicals, fuels, energy and animal feed. We therefore first assessed the variability of the chemical composition of sunflower meal LCF.

Table 5 summarizes the results obtained. The sunflower meal LCF is composed mainly of sunflower hulls and lignocellulosic materials from the stalks, and therefore the LCF is high in fibre and low in protein. Hulls are reported to contain approximately 3-4% crude protein, 2-3.5% fat, 90% crude fiber and 2-5% ash (Cobia & Zimmer, 1978; Kinard, 1981). Due to the


TABLE 3 Essential Amino Acid Con ten t of Sunflower Meal and Enriched L C F (% of

protein) Solvent Extract ion Process

Reference SFM LCF Enriched LCF (96 h cultivation)

FA O a Hen's pattern egg b

Isoleucine 4-0 6'3 4.45 4-26 4-86 Leucine 7.0 8'8 6.45 7"68 6"74 Lysine 5-5 7"0 3.15 3.50 5"03 Meth ion ine - - - - 2.05 2.44 1.95 Cystine 3.5 5.8 4.40 2.95 3.82 Phenyla lanine 3"0 5.7 4'67 3"41 4.36 Tyrosine 3.0 4.2 2-84 4.36 3.90 Threon ine 4.0 5"1 3"59 3-69 4-25 Tryp tophan 1'0 1"5 1-40 1"83 1'70 Valine 5.0 6.8 5.15 5.61 4.95 Arginine - - 6-1 9"16 3'90 7"85 Hist idine - - 2'4 2.20 1.75 1-97

% Prote in - - 12.00 25.10 6"80 22.30

F A O (1973), Gassmann , 1983. h FAO (1970).

TABLE 4 Frac t iona t ion of SFM by F lo ta t ion /Sed imenta t ion at

Different pH Values a

pH 2.0 (%) pH 5.0 (%) pH 7.5 (%)

SFM a 100'0 100'0 100"0 L C F 25'3 __+ 0"6 23"2 ___ 0"6 23"4 ___ 0-3 PF 57-6 ___ 0"3 55"4 ___ 0"4 57"3 _ 0-4 SF b 17"1___0-4 21-4+0"5 19"3+0 '3

a For abbrevia t ions see text. b Calculated as difference: [ 1 0 0 - (LCF + PF)]. Results are average values of three experiments.


TABLE 5 M e a n Chemical Compos i t ion of Sunflower Raw L C F and Enr iched LCF

Component Raw LCF Enriched LCF (%) (96h cultivation) (%)

Dry mat t e r 93.5 ± 0-6 90-9 +_ 0-5 Mois ture 6"5 + 0'5 9.1 + 0.4

Total ni trogen: ( × 5.5) 8-2 + 0.4 - - ( x 6.25) - - 28.9 + 0-7

Protein ni t rogen ( x 5-5) 6"8 _ 0.4 - - ( x 6-25) - - 22"3 + 0"7

Ash 4-3 _ 0'5 8"6 ± 0.4 Fa t 4-8 _ 0-4 4.2 _ 0.2

Acid detergent fibre (ADF) 48-7 + 0-4 22-3 ± 0-4 Neut ra l detergent fibre (NDF) 74.2 ± 0"5 38"8 _ 0"3 Lignin 29'8 + 0"6 11"9 + 0-4 Cellulose 19"9 ± 0'3 10.4 ± 0"3 Hem±cellulose 12"6 ± 0'3 7-2 ± 0"2 Other substnaces 11"9 ± 0"4 4'3 ± 0'3

N F E a 2"0 ± 0-2 4"0 ± 0"4 Soluble sugars 0.2 + 0.1 0.8 ± 0.2 Non-so luble sugars 1-6 ± 0"2 2"7 ± 0"3 Othe r substances 0-2 ± 0.1 0.5 ± 0.2

a N F E = Nitrogen-free extract. All results are expressed on dry matter , and are average values of three experiments.

TABLE 6 Sugars in the Hydrolyzate of Sunflower Meal LCF a

Sugars Percentage of total sugar

Glucose 51.17 ± 1.32 Galactose 20.03 + 0"81 M a n n o s e 4-70 ___ 0.36 Xylanose 21.70 + 0.71 Arab±nose 3.40 _ 0.11

Da ta are the mean of four determinat ions.


TABLE 7 Growth of Trichoderma harzianum S/G2413 on Sunflower

Fermentat ion Source in SSF Meal L C F as

Media Fermentation Protein Increase in time concentration protein concentration (h) (%) (%)

LCF 96 19.4 + 0'5 12"6 _ 0'4 L C F / U - P 96 23-3 __+ 0"8 16'5 _ 0'6 LCF/NH4-P 96 22'0 _ 0'7 15.2 __+ 0"5

Results are average values of three experiments.

difficulty in dehulling oilseeds, sunflower seeds are used undehulled for oil extraction, arid so the sunflower meal LCF derived from oilseed processing is a higher quality product and contains more crude protein (6.8%) and fat (4-8%) and a lower content in fibre (74.2%) than the typical confectionery hull.

The fractional composition of the fibre is presented in Table 5. The main component is lignin (29-8%). Glucose, galactose, mannose, xylose and arabinose were found as products of the enzymatic hydrolysis (Table 6). Glucose is the main sugar.

Growth of Trichoderma harzianum S]G2413 on sunflower meal LCF media in solid-state fermentation

The growth of Trichoderma harzianum S/G2413 on sunflower meal LCF- based media (see Tables 5 and 7) caused an improvement in the protein content (24-28%) after 96 h of cultivation. The higher values were obtained by addition of an exogenous mineral nitrogen source (urea or ammonium sulfate). The additions of an organic nitrogen source such as peptone or NZ- amine were not assayed because of the high commercial costs. The amino acid composition of enriched sunflower meal LCF (see Table 3) showed an important increase in lysine content, from 3.50% to 5.03%, close to the standard values of FAO pattern or Hen's egg protein (FAO, 1970, 1973). The chemical composition of enriched sunflower meal LCF shows a large decrease in fibre content, from 29-8% to 11.9%. All of this, presents sunflower meal LCF as a suitable fermentation source for SSF and the material basically possesses all the exigences of an ideal fermentation source: constant composition for a particular type of oil-extraction process, abundance (c. 200 000 t year- 1), easy to acquire, cheap (US$10-15 t - 1), local (Andalucia, Levante and Castilla), and easy to store.


The widespread production of sunflower oil in some countries suggests the possibility of a real industrial application of sunflower meal-LCF as a

fermentation source.

R E F E R E N C E S

Anon. (1988). Ministerio de Agricultura: Servicio de Estudios de ia Consejeria de Agricultura y Pesca de la Junta de Andalucia, Seviila1988. Boletin no. 8.

Angelova, L. & Dimitrova, M. (1977). Effect of the technology of production on the protein quality of sunflower seed oil meal. 3. Amino acid composition. Nutr. Abstr. Rev., 47, 232.

AOAC (1980). Official Methods of Analysis (13th edn). Association of Official Agricultural Chemists, Washington, DC.

Cheftel, J. C. (1989). In Proteinas Alimentarias. Bioquimica. Propiedades Funcionales. Valor Nutritivo. Modificaciones Quimicas. Ed. J.-C. Cheftel, J. L. Cuq & D. Lorient. Editorial Acribia, S. A., Zaragoza, pp. 107-40.

Cherney, J. H., Volenec, J. J. & Nyquist, W. E. (1985). Sequential fiber analysis of forage as influenced by sample weight. Crop Sci., 25, 1113-15.

Cobia, D. W. & Zimmer, E. D. (eds)(1978). In Sunflower Hulls. Sunflower Production and Marketing. (ND Agricultural Experiment Station, Bulletin No. 25.

Dimitrova, M. (1977). Effect of the technology of production on the quality of protein of sunflower meal. 2. Soluble proteins. Nutr. Abstr. Rev., 47, 223.

Duran, A. & Chereau, D. (1987). A new pilot reactor for solid-state fermentation: application to the protein enrichment of sugar beet pulp. Biotechnol. Bioeng., 31, 476-86.

FAO (1970). Amino Acid Content of Food and Biological Data on Proteins. Nutritional Studies No. 24, FAO, Rome.

FAO (1973). Nutrition Meetings Reports Series no. 52. FAO, Rome. Gassmann, B. (1983). Preparation and application of vegetable proteins, especially

proteins from sunflower seed for human consumption. An approach. Nahrung, 27, 351-69.

Goering, H. K. & van Soest, P. J. (1973). Forage fiber analysis J.A.O.A.C., 4, 1-9. Heredia-Moreno, A., Guillen-BeJarano, R., Fernandez-Bolano, J. & Rivas-

Moreno, M. (1987). Olive stones as a source of fermentable sugars. Biomass, 14, 143-8.

Kinard, D. H. (1981). Feeding Sunflower Products. National Cotton-seed Products Association, Inc., Tennessee 38112. Ref. no. 280.

Parrado, J., Bautista, J. & Machado, A. (1990). Protein enrichment of sunflower meal lignocellulosic fraction by Trichoderma harzianum S/G2413 in solid-state fermentation. Enzyme MicrobioL Technol. (in press).

Smith, D. (1981). Removing and analyzing total non-structural carbohydrates from plant tissue. Wisconsin Agricultural Experiment Station Research Report R2107, University of Wisconsin, Madison, WI, 53706.

Zabriskie, D. W., Armiger, W. B., Phillips, D. H. & Albano, P. A. (1982). Traders' Guide to Fermentation Media Formulation. Traders Proteins, Memphis, TN 38108, p. 14.

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Composition and fractionation of sunflower meal: Use of the lignocellulosic fraction as substrate in solid-state fermentation