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Storage Stability Studies on Pea Flour, Protein Concentrate and Starch
Storage Stability Studies on Pea Flour, Protein Concentrate and Starch A. K. Sumner" L. L. Whalley', G. BlankenageF and e. G. Y oungs' .college of Home Economics, University of Saskatchewan. Saskatoon. Saskatchewan ~[)epartment of Dairy and Food Science, University of ~askatchewan. SaskalOon, Saskatchewan -'Prairie Regional Laboratory, National Research CounCIL Saskatoon. Saskatchewan
Abstract Flour (PF), protein concentrate (PPC) and starch (PS) from field peas (Pisum sativum) were examined for changes during a one year storage period at four moisture levels ranging from 3.8 to 13.6% and temperatures of 7, 21 and 30°C. The standard plate, coliform, yeast and mold and psychrotroph counts usually decreased to less than lO,OOO/g during the storage period. Odor and flavor changes under the most unfavorable storage conditions were probably largely due to lipid changes. Color variations dunng storage appeared to be caused by enzymatJc or non-enzymatic browning near the surface of the glass storage containers.
Resume Les changements dans de la farine (PF), du concentre proteique (PPC), et de l'amidon (PS) de pois des champs (Pisum sativum), au cours d'une annee de stockage, furent examines it quatre niveaux d'humidite allant de 3.8 it 13.6%, et aux temperatures de 7, 21 et 30°C. Ourant cette periode de stockage, on observa habituellement une diminution it moins de 10 OOO/g pour les numerations sur plat de Petri, les coliformes, les levures et moisissures, et les psychrotrophes. Sous les conditions de stockage les plus defavorables, les changements dans l'odeur et le gout furent probablement causes surtout it des changements au niveau des lipides. Les changements dans la couleur au cours du stock age furent apparemment attribuables au brunissement enzymatique ou non-enzymatique pres de la surface des con tenants en verre.
Introduction Field peas are being evaluated as a high protein crop for the Canadian prairies and as a component in agricultural and industrial products. Processes are available for milling the mature field peas into flour which can be classified by wet or dry processes into protein concentrate and starch-rich fractions (Youngs, 1975). These products can be used in foods (PFPS Bulletin No. 1, 1974) and in animal feeds (Bell, 1975) but their wide acceptance requires satisfactory storage properties. Deterioration of grains and their products during storage results from unfavorable conditions which promote microbial and enzymatic activity. The equilibrium relative humidity or water activity of the material determines the availability of moisture for microbial growth (Sallans et al., 1944; Moysey et al., 1975). At a given moisture content the equilibrium relative humidity will vary for different grains and parts of the grain. Each class of microorganism has its characteristic optimum water activity for growth which generally falls in the range of 0.60 to 0.90 (Frazier, 1958). Deterioration during storage can also be caused by lipoxygenase and lipase enzymes which produce undesirable flavors and odors in soy and wheat flours (Mustakas et al., 1969; Daftary et al., 1970; Fellers et al., 1977). The degree of lipid and fatty acid change is enhanced by high moisture and temperature levels. Daftary et al., (1970) showed that deterioration of mold damaged wheat flour was accompanied by the lowering of free and bound lipids. The breakdown of bound lipids was accompanied by a transformation of polar to non-polar-like components Can. Inst. Food Sei. Teehnol. J. Vol. 12. No. 2, April 1979
which might affect functional and bread making qualities of the flour. Color is an important attribute of most flours (Francis et al., 1972). Thus changes which occur during storage such as bleaching can have a marked effect on the product quality. This study investigated changes which occur during storage of pea flour, protein concentrate and starch at moistures up to 13.6% and temperatures as high as 30°e. Emphasis was placed on microbial growth as well as changes in the lipids which could affect flavor and odor properties. Alterations in the product color and functional properties were also analyzed.
Materials and Methods Pea products used in this study were prepared from a ten-lot composite of field peas (Pisum sativum) which included Trapper, Century and Dashaway varieties. Newfield Seeds Limited, Nipawin, Saskatchewan milled the whole peas into flour and air classified some of the flour to produce about one-third as a protein-rich fraction and two thirds as a starch-rich fraction using the dry process described by Y oungs (1975). The proximate composition was determined by standard AOAC (1970) procedures (Table 1). Table 1. Proximate analysis of pea products a .
Analysis Crude Protein %b Crude Lipids % Crude Fibre % Ash % N.F.E. %
Pea Flour 24.5 1.5 6.1 3.2 64.7
Protein Concentrate 54.8 3.0
2.7 5.7 33.8
Starch 3.8 0.4 9.0 1.3
85.5
~Dry basis % N x 6.25
Prior to storage, the flour (PF), protein concentrate (PPC) and starch (PS) were adjusted to four moisture levels within the range 3.8 to 13.6% which encompasses the moisture contents of freshly milled products. Moisture levels were adjusted by humidity, air drying and freeze drying. The samples were placed in triplicate, tightly sealed, sterile Mason jars to simulate sealed moisture- and vaporproof containers. The jars were stored in incubators at 7, 21 and 30°e. Control samples of PF, PPC and PS at the original moisture content were stored at -lO°e. The equilibrium relative humidity was determined for each product with a lithium chloride probe after 33 weeks storage. These values together with the corresponding mois51
ture and temperature were used to prepare graphs for each product which were used later to estimate the equilibrium relative humidity from moisture and temperature measurements. The PF, PPC and PS samples were analyzed for moisture and micro-organisms after 1, 2, 3, 4, 6, 8, 12, 33, and 52 weeks of storage. Total bacteria were determined by carrying out standard plate counts (SPC) on SPC agar after incubation for 3 days at 30°C. Plates for coliform counts, using violet red bile agar, were incubated for 1 day at 32°C. Saborand dextrose agar was used for yeast and mold counts with a plate incubation of 5 days at 21°C and psychrotrophs were enumerated on SPC agar after 10 days at 7°C. After 52 weeks of storage, samples were plated with violet red bile agar and plates were incubated at 45°C for 24 hr. Any visible colony at this temperature was assumed to be E. coli. Samples were screened for aflatoxin using the AOAC (1970) method for peanut products. Lipid analysis was carried out by extraction of the samples for 16 hr. in a soxhlet using diethylether containing n-docosanoic acid ( C 22:0) as an internal standard. Total fatty acids were determined as methyl esters by saponification with methanolic KOH and esterification using BF:,-etherate as an esterification catalyst. The methyl esters were dissolved in CS 2 and injected into a Hewlett-Packard Model 7620A gas chromatograph with flame ionization detector. The stainless steel column was 8 ft x Vs in. o.d. packed with 1% ethylene glycol succinate/4% butanediol succinate on 60/80 A W Chromosorb W. Operating temperatures were as follows: column, 200°C; injection port, 230°C; FID detector 230°C. The helium carrier gas flow rate was 30 mllmin. Free fatty acids in the diethylether extract were esterified with diazomethane and analyzed in the gas chromatograph as described previously. Color was determined by measuring the absorption on an Hitachi Perkin Elmer 139, UV-VIS spectrophotometer with a reflectance attachment. The water absorption and nitrogen solubility index (NSI) were analyzed on stored and control samples. Combined methods of SoUars (1972) and Sosulski (1962) were used to measure water absorption at natural pH 6.6. A 5 g sample was shaken with 30 ml water in a 50 ml centrifuge tube at No. 10 speed on a Burrell shaker. After centrifuging at 1200 x g for 25 min, the supernatant was decanted and the tube drained at a 45 degree angle for 10 min. The retained water was determined by weighing and expressed as a per cent of the original sample weight. The NSI method was based on AOCS (1974) procedure except for the extraction. This was carried out in a Blue M MagniWhirl constant temperature bath at 30°C where the sample was shaken at 120 rev Imin for 2 hr. The NSl was determined at pH 3.9 and 6.6 for PF and 3.9 for PPc. The pH of the aqueous solution was adjusted with predetermined quantities of IN HCl or IN NaOH before addmg the sample, then checked 5 min after sample addition when a final adjustment was made if required. The flavor of the stored pea flour samples was compared to a control by a panel of 9 judges who were served a 0.5% aqueous slurry in 20 ml plastic creamers with straws and fOil lids to prevent color bias or odor loss. 52
Results and Discussion The equilibrium relative humidity of the stored sam_ ples varied from less than 10% to a maximum of 67% for pea flour depending on the temperature, moisture content and product. The composition of the stored material and the physical structure of the product both affect the equili_ brium relative humidity. (Colvin et al., 1947; Moyseyet al., 1975). Microbial counts for pea products decreased sharply by the end of the one year storage period (Figures I, 2, 3).
6
•
7°C, 61% R.H.
o
21°C, 65% R.H.
• 30°C, 67% R.H.
:!!
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Cl<
~ u
5
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o Fig. 1.
10
20
30 40 TIME, WEEKS
50
60
70
Effect of storage temperature on standard plate count for pea flour at 13.5% moisture over a one year storage period.
Even the highest equilibrium relative humidity of 67% appeared to be too low to sustain growth. This is consistent with the observations of others who report that a water activity at this level is unlikely to cause spoilage of foods stored at room temperature (Frazier, 1958). Local areas of high humidity may occur due to moisture transfer resulting from temperature differences (Christens en et al., 1972). Temperature, because of its relationship to relative humidity, had a mjor effect on the SPC of pea flour containing 13.5% moisture (Figure 1). The initial count of 440,000/g decreased to less than 1O,OOO/g in 12 weeks at 30°C and in 52 weeks at 21°C. At rc the microbial count only decreased to 220,OOO/g in 52 weeks. Decreased populations occurred most rapidly at the higher temperatures probably due to the more rapid development of unfavorable conditions such as the accumulation of metabolic products. Moisture content also influenced the SPC of pea flour stored at 30°C because it affected the product's relative humidity as shown in Figure 2. The equilibrium moisture contents of 5.2, 8.2, 10.2 and 13.5% corresponded to equilibrium relative humidities ranging from 10 to 67%. The moisture content of the samples remained constant during the 52 week storage period except for the two highest J. Inst. Can. Sci. Technol. Aliment. Vol. 12, No. 2, Avril 1979
Table 2. Fatty acid analysis a of pea products during one year storage. PEA FLOUR - 13% moisture, 30°C FA as % of Total FA FA as % of FFA Control b Control c 33 33 52 12
Storage - weeks Fatty acid - % Palmitic 16:0 Stearic 18:0 Oleic 18:1 Linoleic 18:2 18:3 Linolenic
12.9 3.1 22.0 48.6 13.4
10.3 1.7 30.0 53.6 4.4
11.5 2.5 28.9 52.4 4.7
12.6 3.5 22.0 48.7 13.2
PEA PROTEIN CONCENTRATE FA as % of Total FA 33 52 Control b
Storage - weeks Fatty acid - % Palmitic 16:0 18:0 Stearic Oleic 18:1 Linoleic 18:2 Linolenic 18:3
12.8 3.1 23.0 48.4 12.7
9.9 2.4 26.0 52.7 9.0
Control c
12.3 1.9 25.8 51.5 8.5
FA as % of Total FA 33 52 Control b
Storage - weeks Fatty acid - % Palmitic 16:0 Stearic 18:0 Oleic 18:1 Linoleic 18:2 Linolenic 18:3
13.0 3.7 21.4 46.9 15.0
FA as % of FFA 12 33 11.1 3.2 24.6 52.2 8.9
13.3 3.5 22.7 48.8 1l.7
52 13.6 2.4 26.8 48.9 8.3
13% moisture, 30°C
Control c
14.7 2.3 25.1 48.9 9.0
14.2 3.4 24.5 47.5 10.4
16.2 2.8 34.7 43.9 2.4
13% moisture, 30°C
12.6 3.5 22.8 48.4 12.7
PEA STARCH -
16.6 3.3 29.6 46.4 4.1
12.0 3.4 23.4 50.3 10.9
52
FA as % of FFA 12 33
13.5 4.0 21.9 47.0 13.6
14.0 3.2 23.5 47.6 11.7
13.8 4.0 23.4 46.8 12.0
52 12.9 2.3 24.2 49.2 11.4
aRelative fatty acid distribution on weight % basis. bDetermined after 52 weeks storage at -10°C. CDetermined after 33 weeks storage at -10°C
l.lr----------------------,
1.1 •
Control
o 33 weeks 0.9
...u
0.9
o 52 weeks
0.7
...u
0.5
V'I cO
Z
Z
cO 0<
cO 0<
o
0
V'I
cO
0.7
0.5 • Control
0.3
o 33 Weeks
0.3
o 52 Weeks 0.1
L--_ _-'--_ _--'-_ _- ' -_ _--L_ _---lL-_----I
350
400
450
500
WAVELENGTH,
550
600
650
mj.l
0.1
~
350
__
~
__
400
~
_ _ ___'__ _
450
500
WAVELENGTH,
~L_
550
_ _ _ L_ _ _ J
600
650
mj.4
*Psychrotrophs were counted on samples stored at 7°C. Light absorbance of pea flour stored at 13% moisture and 30°C for one year.
Fig. 5 Light absorbance of pea protein concentrate stored at 13.6% moisture and 30°C for one year.
They occurred near the surface. of the storage bottles where condensation could cause Increased water content and water activity. Under these conditions deterioration may occur due to enzymatic or non-enzymatic browning
(Labuza et al., 1972). Enzymatic browning may result from the oxidation of compounds such as polyphenols. Non-enzymatic browning such as carbonyl-amine reactions occur over a wide range of relative humidities. Labuza et al.
Fig. 4
54
J. Inst. Can. Sci. Technol. Aliment. Vol. 12, No. 2, Avril 1979
(1970) reported that the non-enzymatic browning rate for a soup mix was at a maximum between 60% and 80% relative humidities. The PF and PPC storage samples which showed color changes, also had relative humidities within this range and probably contained more enzymes and non-enzymatic .~rO\yning reactants than PS. . Nitrogen solublhty mdex (NSI) and water absorptIOn properties were not adversely affected during storage. The water absorption of PF and PPC stored under the most severe conditions was 98% and 86% respectively compared to 96% and 86% for control samples stored at -lODe. There was little or no change in the NSI of PF and PPC during the storage period as shown by the values for the stored and control samples (Table 3). This suggests Table 3. Nitrogen solubility index of pea products after one year storage. Sample
Pea flour - control Pea flour - stored Pea flour - control Pea flour - stored Protein Conc.--control Protein Conc.~stored
Moisture %
Storage Temp. QC
NSI Extraction pH
NSI %
13.5 13.5 \3.5 13.5 13.6 13.6
-10 30 -10 30 -10 30
3.9 3.9 6.6 6.6 3.9 3.9
12 12 82 83 17 17
that there was no major physical or chemical changes in the protein during storage. The lower values at pH 3.9 compared to pH 6.5 resulted from the samples being close to the protein isoelectric point of about pH 4.5 where nitrogen solubility is at a minimum. The NSI results agree quite closely with the NSI values reported in PFPS Bulletin No. 1 (1974). The odor of PF and PPC samples stored at the two highest moisture levels and 30°C changed from a fresh pea odor at the beginning of the trial to a musty, fishy odor. Samples stored at less than 10% moisture and 30°C did not develop the unpleasant odors. Mustiness is usually caused by microbial growth while undesirable odors and flavors are attributed to enzymatic modifications of the lipids (Haydar et al., 1975; Hinchc1iffe et al., 1977). The unpleasant odors were volatile and disappeared rapidly. An untrained panel of 9 compared the flavor of a control pea flour with samples which were stored under various moisture and temperature conditions for one year (Table 4). None of the samples were judged to be significantly different (P < 0.5) from the control which was stored at -lODe. More panelists detected flavor changes Table 4. Flavor evaluation of pea flour stored for one year. Flour Storage Conditions Moisture % Temp. QC 13.5 13.5 13.5 10.2 8.2 5.2 5.2 11.3
No. of panelists judging sample differs from control a
30 21
7 30 30 30 7 -10
6
5 2 3
4 3 2 Control
aOn1y significant (P <0.05) for judgements of 8 or more. Can. Inst. Food Sei. Teehnol. J. Vol. 12, No. 2, April 1979
under the severe conditions of 13.5% moisture and storage temperatures of 21°C and 30°e. Panelists who detected flavor changes correctly described them by such varied terms as mild, musty, rancid and bitter. Similarly, fababean flour was reported to develop a bitter flavor after storage for one year at ambient temperature (Hinchcliffe et al. (1977), Fellers et aI, (1977) reported that the flavor and odor stability of flours could be enhanced greatly by small moisture decreases below 13% when the products were stored at lOO°F (37.8°C).
Conclusions Moisture content and temperature are major factors affecting the storage stability of pea products, As the resulting relative humidity increases, there is a greater possibility of microbial spoilage, Even if microbial populations eventually decrease, their metabolic products may cause product deterioration through odor and flavor changes, Enzymatic and chemical changes may also affect the products during storage, A maximum equilibrium relative humidity of 60% should provide satisfactory product stability under many conditions. However, safe levels of moisture and equilibrium relative humidity can only be established after further investigation of such factors as temperature cycling and storage in commercial shipping containers,
Acknowledgements Financial support for this research was provided by the Saskatchewan Research Council and the Canada Agriculture New Crop Development Fund, The technical assistance of Dr. M, Prior, Canada Agriculture for aflatoxin analysis, Mr. L. Hoffman, Prairie Regional Laboratory for fatty acid analysis and Mrs, Marilyn Nielsen is gratefully acknowledged,
References AOAC 1970. Official Methods of Analvsis. 11th ed. Assoc. ofOffic. Agric. Chemists. Wash .. D.e. AOeS. 1974. Official and Tentative Methods of the American Oil Chemists Societv. Third Edit. Amer Oil Chem. Soc. Champaign. Ill. .. . . Bell,1. M. 1975. Utilization of protein supplements m alllmai feeds. In Harapwk, J. T. (Ed.) Oilseed and Pulse Crops in Western Canada-A Symposium. Western Co-op. Fertilizers Ltd., Calgary, Alberta. Christensen. C. M. and Kaufman, H. H. 1972. Biological processes in stored soybeans. In Soybeans: Chemistry and Technology. Vol. 1. Avi Pub. Co .. Westpon, Conn. Colvin. R .. Craig. B. M. and Sallans. H. R. 1947. Hygroscopic equilibria for hulls and kernels of sunflowerseed and oats. Can. J. Research F, 25: Ill. Daftarv. R. D., PomeranL Y. and Sauer. D. B. 1970. Changes in wheat flour damaged by maid during storage. Effects of lipid, lipoprotein and protein. J. Agric. Food Chem .. 18:613 Fellers. D. A. and Bean, M. M. 1977. Storage stability of wheat based foods-A revIew. J. Food SeL 42: [[43. Francis, F. 1. and Clydesdale, F. M. 1972 Color measurement of foods. Part IV Cereal products. Food Prod. Dev. 6:(4)34. Frazier. W. C. 1958. Food Microhiology. McGra",:,-Hill, New York. . Haydar. M .. Steele. L.. and Hadziyev. D. 1975. OXIdation of pea liplds hy pea secd hpoxygcnase. 1. Food Sci. 40:808 Hinchc!iffe, C. Mc DanieL M., Vaisev, M .. and [skin, N. A. M. 1977. The Havor of fabaheam as affected by heat and storage. C"an. lnst. Food Sei. Teehnol. J. 10: 181. Labuza. T. P., Tannenbaum, S. R. and KareL M. 1970. Water content and stability or low-moisture and intermediate-moisture foods. Food Technol. 24:35. Labuza. T. P., MeN ally, L, Gallagher, D .. Hawkes, J. and Hurtado. F. 1972. Stability of intermediate moisture "foods. J. Food Sci. 37: 154. Moysey. E. B. and Norum, E. R. 1975. Storage. drying and handling of oilseeds and pulse crops In Harapiak, J. T. (Ed.) Oilseed and Pulse Crops in Western Canada-A SymposIum. Western Co-op. Fertilit-ers Ltd .. Calgary. Alberta. Mustakas. G. C. A[breeht, W. 1., McGhee. J. E.. Black. L. T, Bookwalter. G N. Griffin. E. L 1969. Lipoxidase deactivation 10 improve soluhility. odor and flaw)f of full-fat soy' flour. J. Amer. OiL Chem. Soc. 46:623. PFPS Bulletin No. 1, 1974. Pea flour and pea protein concentrate. Prep~~red by the Prairie Regional Laboratory. Nat. Res. Council and College of Home EconomIcs, Univ. of Sask.. Saskatoon, Sask. Sallans. H. R., Sinclair. G. D. and Larmour. R. K. 1944. The spontaneous heating of flaxseed and sunflower seeds stored under adiabatic conditions. Can. J. Res .. F. 22: 18t. Sollars. W. F. 1972. Relation of distilled-water retention [0 alkaline-water retention, water absorption. and baking properties of wheat flour. Cereal Chem. 49: 168. Sosulski. F. W. 1962. The centrifuge method for determining flour absorption in hard red spring wheat". Cereal Chem. 39:344. Youngs. C. G. 1975. Primary processing of pulse. InHarapiak. J: T. (Ed.) Oilseed and Pulse Crops in Western Canada-A Symposium. Western Co-op. Ferttlizers Ltd .. Calgary. Alberta. Received June 22, 1978
55
Abstract Flour (PF), protein concentrate (PPC) and starch (PS) from field peas (Pisum sativum) were examined for changes during a one year storage period at four moisture levels ranging from 3.8 to 13.6% and temperatures of 7, 21 and 30°C. The standard plate, coliform, yeast and mold and psychrotroph counts usually decreased to less than lO,OOO/g during the storage period. Odor and flavor changes under the most unfavorable storage conditions were probably largely due to lipid changes. Color variations dunng storage appeared to be caused by enzymatJc or non-enzymatic browning near the surface of the glass storage containers.
Resume Les changements dans de la farine (PF), du concentre proteique (PPC), et de l'amidon (PS) de pois des champs (Pisum sativum), au cours d'une annee de stockage, furent examines it quatre niveaux d'humidite allant de 3.8 it 13.6%, et aux temperatures de 7, 21 et 30°C. Ourant cette periode de stockage, on observa habituellement une diminution it moins de 10 OOO/g pour les numerations sur plat de Petri, les coliformes, les levures et moisissures, et les psychrotrophes. Sous les conditions de stockage les plus defavorables, les changements dans l'odeur et le gout furent probablement causes surtout it des changements au niveau des lipides. Les changements dans la couleur au cours du stock age furent apparemment attribuables au brunissement enzymatique ou non-enzymatique pres de la surface des con tenants en verre.
Introduction Field peas are being evaluated as a high protein crop for the Canadian prairies and as a component in agricultural and industrial products. Processes are available for milling the mature field peas into flour which can be classified by wet or dry processes into protein concentrate and starch-rich fractions (Youngs, 1975). These products can be used in foods (PFPS Bulletin No. 1, 1974) and in animal feeds (Bell, 1975) but their wide acceptance requires satisfactory storage properties. Deterioration of grains and their products during storage results from unfavorable conditions which promote microbial and enzymatic activity. The equilibrium relative humidity or water activity of the material determines the availability of moisture for microbial growth (Sallans et al., 1944; Moysey et al., 1975). At a given moisture content the equilibrium relative humidity will vary for different grains and parts of the grain. Each class of microorganism has its characteristic optimum water activity for growth which generally falls in the range of 0.60 to 0.90 (Frazier, 1958). Deterioration during storage can also be caused by lipoxygenase and lipase enzymes which produce undesirable flavors and odors in soy and wheat flours (Mustakas et al., 1969; Daftary et al., 1970; Fellers et al., 1977). The degree of lipid and fatty acid change is enhanced by high moisture and temperature levels. Daftary et al., (1970) showed that deterioration of mold damaged wheat flour was accompanied by the lowering of free and bound lipids. The breakdown of bound lipids was accompanied by a transformation of polar to non-polar-like components Can. Inst. Food Sei. Teehnol. J. Vol. 12. No. 2, April 1979
which might affect functional and bread making qualities of the flour. Color is an important attribute of most flours (Francis et al., 1972). Thus changes which occur during storage such as bleaching can have a marked effect on the product quality. This study investigated changes which occur during storage of pea flour, protein concentrate and starch at moistures up to 13.6% and temperatures as high as 30°e. Emphasis was placed on microbial growth as well as changes in the lipids which could affect flavor and odor properties. Alterations in the product color and functional properties were also analyzed.
Materials and Methods Pea products used in this study were prepared from a ten-lot composite of field peas (Pisum sativum) which included Trapper, Century and Dashaway varieties. Newfield Seeds Limited, Nipawin, Saskatchewan milled the whole peas into flour and air classified some of the flour to produce about one-third as a protein-rich fraction and two thirds as a starch-rich fraction using the dry process described by Y oungs (1975). The proximate composition was determined by standard AOAC (1970) procedures (Table 1). Table 1. Proximate analysis of pea products a .
Analysis Crude Protein %b Crude Lipids % Crude Fibre % Ash % N.F.E. %
Pea Flour 24.5 1.5 6.1 3.2 64.7
Protein Concentrate 54.8 3.0
2.7 5.7 33.8
Starch 3.8 0.4 9.0 1.3
85.5
~Dry basis % N x 6.25
Prior to storage, the flour (PF), protein concentrate (PPC) and starch (PS) were adjusted to four moisture levels within the range 3.8 to 13.6% which encompasses the moisture contents of freshly milled products. Moisture levels were adjusted by humidity, air drying and freeze drying. The samples were placed in triplicate, tightly sealed, sterile Mason jars to simulate sealed moisture- and vaporproof containers. The jars were stored in incubators at 7, 21 and 30°e. Control samples of PF, PPC and PS at the original moisture content were stored at -lO°e. The equilibrium relative humidity was determined for each product with a lithium chloride probe after 33 weeks storage. These values together with the corresponding mois51
ture and temperature were used to prepare graphs for each product which were used later to estimate the equilibrium relative humidity from moisture and temperature measurements. The PF, PPC and PS samples were analyzed for moisture and micro-organisms after 1, 2, 3, 4, 6, 8, 12, 33, and 52 weeks of storage. Total bacteria were determined by carrying out standard plate counts (SPC) on SPC agar after incubation for 3 days at 30°C. Plates for coliform counts, using violet red bile agar, were incubated for 1 day at 32°C. Saborand dextrose agar was used for yeast and mold counts with a plate incubation of 5 days at 21°C and psychrotrophs were enumerated on SPC agar after 10 days at 7°C. After 52 weeks of storage, samples were plated with violet red bile agar and plates were incubated at 45°C for 24 hr. Any visible colony at this temperature was assumed to be E. coli. Samples were screened for aflatoxin using the AOAC (1970) method for peanut products. Lipid analysis was carried out by extraction of the samples for 16 hr. in a soxhlet using diethylether containing n-docosanoic acid ( C 22:0) as an internal standard. Total fatty acids were determined as methyl esters by saponification with methanolic KOH and esterification using BF:,-etherate as an esterification catalyst. The methyl esters were dissolved in CS 2 and injected into a Hewlett-Packard Model 7620A gas chromatograph with flame ionization detector. The stainless steel column was 8 ft x Vs in. o.d. packed with 1% ethylene glycol succinate/4% butanediol succinate on 60/80 A W Chromosorb W. Operating temperatures were as follows: column, 200°C; injection port, 230°C; FID detector 230°C. The helium carrier gas flow rate was 30 mllmin. Free fatty acids in the diethylether extract were esterified with diazomethane and analyzed in the gas chromatograph as described previously. Color was determined by measuring the absorption on an Hitachi Perkin Elmer 139, UV-VIS spectrophotometer with a reflectance attachment. The water absorption and nitrogen solubility index (NSI) were analyzed on stored and control samples. Combined methods of SoUars (1972) and Sosulski (1962) were used to measure water absorption at natural pH 6.6. A 5 g sample was shaken with 30 ml water in a 50 ml centrifuge tube at No. 10 speed on a Burrell shaker. After centrifuging at 1200 x g for 25 min, the supernatant was decanted and the tube drained at a 45 degree angle for 10 min. The retained water was determined by weighing and expressed as a per cent of the original sample weight. The NSI method was based on AOCS (1974) procedure except for the extraction. This was carried out in a Blue M MagniWhirl constant temperature bath at 30°C where the sample was shaken at 120 rev Imin for 2 hr. The NSl was determined at pH 3.9 and 6.6 for PF and 3.9 for PPc. The pH of the aqueous solution was adjusted with predetermined quantities of IN HCl or IN NaOH before addmg the sample, then checked 5 min after sample addition when a final adjustment was made if required. The flavor of the stored pea flour samples was compared to a control by a panel of 9 judges who were served a 0.5% aqueous slurry in 20 ml plastic creamers with straws and fOil lids to prevent color bias or odor loss. 52
Results and Discussion The equilibrium relative humidity of the stored sam_ ples varied from less than 10% to a maximum of 67% for pea flour depending on the temperature, moisture content and product. The composition of the stored material and the physical structure of the product both affect the equili_ brium relative humidity. (Colvin et al., 1947; Moyseyet al., 1975). Microbial counts for pea products decreased sharply by the end of the one year storage period (Figures I, 2, 3).
6
•
7°C, 61% R.H.
o
21°C, 65% R.H.
• 30°C, 67% R.H.
:!!
«
Cl<
~ u
5
Q. V>
o Fig. 1.
10
20
30 40 TIME, WEEKS
50
60
70
Effect of storage temperature on standard plate count for pea flour at 13.5% moisture over a one year storage period.
Even the highest equilibrium relative humidity of 67% appeared to be too low to sustain growth. This is consistent with the observations of others who report that a water activity at this level is unlikely to cause spoilage of foods stored at room temperature (Frazier, 1958). Local areas of high humidity may occur due to moisture transfer resulting from temperature differences (Christens en et al., 1972). Temperature, because of its relationship to relative humidity, had a mjor effect on the SPC of pea flour containing 13.5% moisture (Figure 1). The initial count of 440,000/g decreased to less than 1O,OOO/g in 12 weeks at 30°C and in 52 weeks at 21°C. At rc the microbial count only decreased to 220,OOO/g in 52 weeks. Decreased populations occurred most rapidly at the higher temperatures probably due to the more rapid development of unfavorable conditions such as the accumulation of metabolic products. Moisture content also influenced the SPC of pea flour stored at 30°C because it affected the product's relative humidity as shown in Figure 2. The equilibrium moisture contents of 5.2, 8.2, 10.2 and 13.5% corresponded to equilibrium relative humidities ranging from 10 to 67%. The moisture content of the samples remained constant during the 52 week storage period except for the two highest J. Inst. Can. Sci. Technol. Aliment. Vol. 12, No. 2, Avril 1979
Table 2. Fatty acid analysis a of pea products during one year storage. PEA FLOUR - 13% moisture, 30°C FA as % of Total FA FA as % of FFA Control b Control c 33 33 52 12
Storage - weeks Fatty acid - % Palmitic 16:0 Stearic 18:0 Oleic 18:1 Linoleic 18:2 18:3 Linolenic
12.9 3.1 22.0 48.6 13.4
10.3 1.7 30.0 53.6 4.4
11.5 2.5 28.9 52.4 4.7
12.6 3.5 22.0 48.7 13.2
PEA PROTEIN CONCENTRATE FA as % of Total FA 33 52 Control b
Storage - weeks Fatty acid - % Palmitic 16:0 18:0 Stearic Oleic 18:1 Linoleic 18:2 Linolenic 18:3
12.8 3.1 23.0 48.4 12.7
9.9 2.4 26.0 52.7 9.0
Control c
12.3 1.9 25.8 51.5 8.5
FA as % of Total FA 33 52 Control b
Storage - weeks Fatty acid - % Palmitic 16:0 Stearic 18:0 Oleic 18:1 Linoleic 18:2 Linolenic 18:3
13.0 3.7 21.4 46.9 15.0
FA as % of FFA 12 33 11.1 3.2 24.6 52.2 8.9
13.3 3.5 22.7 48.8 1l.7
52 13.6 2.4 26.8 48.9 8.3
13% moisture, 30°C
Control c
14.7 2.3 25.1 48.9 9.0
14.2 3.4 24.5 47.5 10.4
16.2 2.8 34.7 43.9 2.4
13% moisture, 30°C
12.6 3.5 22.8 48.4 12.7
PEA STARCH -
16.6 3.3 29.6 46.4 4.1
12.0 3.4 23.4 50.3 10.9
52
FA as % of FFA 12 33
13.5 4.0 21.9 47.0 13.6
14.0 3.2 23.5 47.6 11.7
13.8 4.0 23.4 46.8 12.0
52 12.9 2.3 24.2 49.2 11.4
aRelative fatty acid distribution on weight % basis. bDetermined after 52 weeks storage at -10°C. CDetermined after 33 weeks storage at -10°C
l.lr----------------------,
1.1 •
Control
o 33 weeks 0.9
...u
0.9
o 52 weeks
0.7
...u
0.5
V'I cO
Z
Z
cO 0<
cO 0<
o
0
V'I
cO
0.7
0.5 • Control
0.3
o 33 Weeks
0.3
o 52 Weeks 0.1
L--_ _-'--_ _--'-_ _- ' -_ _--L_ _---lL-_----I
350
400
450
500
WAVELENGTH,
550
600
650
mj.l
0.1
~
350
__
~
__
400
~
_ _ ___'__ _
450
500
WAVELENGTH,
~L_
550
_ _ _ L_ _ _ J
600
650
mj.4
*Psychrotrophs were counted on samples stored at 7°C. Light absorbance of pea flour stored at 13% moisture and 30°C for one year.
Fig. 5 Light absorbance of pea protein concentrate stored at 13.6% moisture and 30°C for one year.
They occurred near the surface. of the storage bottles where condensation could cause Increased water content and water activity. Under these conditions deterioration may occur due to enzymatic or non-enzymatic browning
(Labuza et al., 1972). Enzymatic browning may result from the oxidation of compounds such as polyphenols. Non-enzymatic browning such as carbonyl-amine reactions occur over a wide range of relative humidities. Labuza et al.
Fig. 4
54
J. Inst. Can. Sci. Technol. Aliment. Vol. 12, No. 2, Avril 1979
(1970) reported that the non-enzymatic browning rate for a soup mix was at a maximum between 60% and 80% relative humidities. The PF and PPC storage samples which showed color changes, also had relative humidities within this range and probably contained more enzymes and non-enzymatic .~rO\yning reactants than PS. . Nitrogen solublhty mdex (NSI) and water absorptIOn properties were not adversely affected during storage. The water absorption of PF and PPC stored under the most severe conditions was 98% and 86% respectively compared to 96% and 86% for control samples stored at -lODe. There was little or no change in the NSI of PF and PPC during the storage period as shown by the values for the stored and control samples (Table 3). This suggests Table 3. Nitrogen solubility index of pea products after one year storage. Sample
Pea flour - control Pea flour - stored Pea flour - control Pea flour - stored Protein Conc.--control Protein Conc.~stored
Moisture %
Storage Temp. QC
NSI Extraction pH
NSI %
13.5 13.5 \3.5 13.5 13.6 13.6
-10 30 -10 30 -10 30
3.9 3.9 6.6 6.6 3.9 3.9
12 12 82 83 17 17
that there was no major physical or chemical changes in the protein during storage. The lower values at pH 3.9 compared to pH 6.5 resulted from the samples being close to the protein isoelectric point of about pH 4.5 where nitrogen solubility is at a minimum. The NSI results agree quite closely with the NSI values reported in PFPS Bulletin No. 1 (1974). The odor of PF and PPC samples stored at the two highest moisture levels and 30°C changed from a fresh pea odor at the beginning of the trial to a musty, fishy odor. Samples stored at less than 10% moisture and 30°C did not develop the unpleasant odors. Mustiness is usually caused by microbial growth while undesirable odors and flavors are attributed to enzymatic modifications of the lipids (Haydar et al., 1975; Hinchc1iffe et al., 1977). The unpleasant odors were volatile and disappeared rapidly. An untrained panel of 9 compared the flavor of a control pea flour with samples which were stored under various moisture and temperature conditions for one year (Table 4). None of the samples were judged to be significantly different (P < 0.5) from the control which was stored at -lODe. More panelists detected flavor changes Table 4. Flavor evaluation of pea flour stored for one year. Flour Storage Conditions Moisture % Temp. QC 13.5 13.5 13.5 10.2 8.2 5.2 5.2 11.3
No. of panelists judging sample differs from control a
30 21
7 30 30 30 7 -10
6
5 2 3
4 3 2 Control
aOn1y significant (P <0.05) for judgements of 8 or more. Can. Inst. Food Sei. Teehnol. J. Vol. 12, No. 2, April 1979
under the severe conditions of 13.5% moisture and storage temperatures of 21°C and 30°e. Panelists who detected flavor changes correctly described them by such varied terms as mild, musty, rancid and bitter. Similarly, fababean flour was reported to develop a bitter flavor after storage for one year at ambient temperature (Hinchcliffe et al. (1977), Fellers et aI, (1977) reported that the flavor and odor stability of flours could be enhanced greatly by small moisture decreases below 13% when the products were stored at lOO°F (37.8°C).
Conclusions Moisture content and temperature are major factors affecting the storage stability of pea products, As the resulting relative humidity increases, there is a greater possibility of microbial spoilage, Even if microbial populations eventually decrease, their metabolic products may cause product deterioration through odor and flavor changes, Enzymatic and chemical changes may also affect the products during storage, A maximum equilibrium relative humidity of 60% should provide satisfactory product stability under many conditions. However, safe levels of moisture and equilibrium relative humidity can only be established after further investigation of such factors as temperature cycling and storage in commercial shipping containers,
Acknowledgements Financial support for this research was provided by the Saskatchewan Research Council and the Canada Agriculture New Crop Development Fund, The technical assistance of Dr. M, Prior, Canada Agriculture for aflatoxin analysis, Mr. L. Hoffman, Prairie Regional Laboratory for fatty acid analysis and Mrs, Marilyn Nielsen is gratefully acknowledged,
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