2Department of Human Nutrition and Home Economics, Box 1, Kyambogo University Uganda
Key Words: UNImix porridge, Trypsin inhibitors, Haemagglutinins, LAB Uji culture and Lactic fermentation.
Results from investigations pointed to inadequate inactivation/elimination of antitrypsin and hemagglutinin factors in the UNImix porridge containing kidney beans, and thus established grounds for the development of the present day UNImix porridge which contains soybeans instead of kidney beans and is extruded-cooked to inactivate trypsin inhibitors and haemagglutinins in the soybeans [1].
Since then several therapeutic porridge rations and complementary foods have been developed along the porridge concept for nutritional intervention and rehabilitation such as WFP mix by the World food program and Corn Soy Blend (CSB) from USA by USAID. The use of porridge rations for nutritional intervention and rehabilitation programs across Sub-Saharan region of Africa in refugee camps, famine distress spots and with people living with HIV virus has been a great success in countries such as Somalia, Kenya, Uganda, Democratic Republic of Congo (DRC), Ethiopia, South Sudan, Darfur region of Sudan, Malawi, Mozambique and Angola [2].
However, Nutritionists in Darfur region of Sudan reported cases of flatulence and diarrhea, including reduced UNImix porridge intake by children due to its low palatability. Flatulence and diarrhea can occur in some individuals caused by alphaoligosaccharides namely raffinose, verbascose and stachyose found in legumes like soybeans. Unlike Trypsin inhibitors (Ti) and haemagglutinins (Hgg) also found in legumes, alphaolygosaccharides cannot be eliminated by extrusion-cooking in production of UNImix porridge [3]. The alpha-oligosaccharides can however be eliminated by lactic fermentation through hydrolysis using appropriate Lactic Acid Bacteria (LAB) strains such as in Uji starter culture reported to also inactivate trypsin inhibitors and hemagglutinins [4]. The Uji culture exhibited invitro antimicrobial properties against some diarrheal causing pathogens but not insitu [8]. Several strains of Lactobacillus plantarum strains and especially those studied in some fermented milk products and porridges have been shown to have some probiotic properties.
Lactic fermentation can simultaneously impart several other beneficial nutritional functionalities in UNImix porridge such as improved palatability due to developed appealing taste and flavors, cook ability, protein and starch digestibility, minerals’ bioavailability, lowered dietary bulk, and antimicrobial properties [5-10]. Minerals’ bioavailability is made possible by LAB strains with phytase activity, while improved palatability increases porridge intake critical for enhancing convalescence among clinically malnourished children [3, 10 -12].
This study was therefore aimed at substituting extrusioncooking with lactic fermentation in UNImix porridge production, with special regard to inactivation/elimination of trypsin inhibitors and hemagglutinins using Uji (LAB) starter culture dominated with strains of Lactobacillus plantarum, some strains of Pediococcus acidilactici and Pediococcus pentosaceus known to eliminate by hydrolysis alpha-oligosaccharides and improve palatability of porridges [4, 5].
Maize, Soya bean and Kidney bean flours were obtained from Proctor and Allan Co. Ltd Nairobi, while Soybeans and flakes were obtained from Soy Afric.Co. Ltd Nairobi, having been imported from Ohio, U.S.A. The two companies were contracted by UNICEF Somalia to manufacture UNImix porridge ration.
Fermentation of Unimix
Slurry samples of UNImix porridge were fermented using Uji starter culture prepared according to Mbugua 1992 [13]. Optimal conditions used for fermentation were ambient temperatures ranging from about 22oC -35oC in keeping with traditional fermentation. Uji culture a mesophilic starter culture was developed, from the traditional fermentation process, and reduced traditional fermentation time from about 72 to 24 hrs.
Fermented slurry samples were prepared by mixing flour blends of UNImix with tap water at 40% w/w flour solids, inoculating with Uji starter culture and incubating at 25oC for 24 hrs in a 3 × 5 factorial design, according to Figure 1. Fermented samples: K1 – K5, S1 – S5 and Sf1 – Sf5, were analysed for presence of Trypsin Inhibitors (Ti) and Haemagglutinins (Hgg) without boiling and after boiling for 3, 5, 10, and 15 minutes.
Boiling of Samples
The fermented UNImix samples with 40% flour solids were diluted five times to 8% solids in 125 ml volumes in aluminum cups measuring 6.2 and 7.0 cm of diameter and height respectively and with lids. This helped achievement of appropriate consistency and viscosity for consumption. The slurry samples were brought to boil on a hot plate while stirring, covered and allowed to simmer for 3, 5, 10, and 15 minutes and cooled to 25oC before analysis for Ti and Hgg. The boiling temperature for the slurries was 93oC, the boiling and cooking temperature in Nairobi due to the attitude, but higher or lower temperatures due attitudes would not matter as long as temperatures do not drop below starch gelatinisation of 72oC only possible in inhabitable high mountains.
Detection of Trypsin Inhibitors (Ti)
Trypsin inhibitors were detected by a reaction between crude protein extract from slurry samples and gelatin in the presence of trypsin enzyme according to Reddy et al 1986 [3]. The crude protein was extracted from samples by mixing 5g of slurry with 100ml of 0.1N sodium acetate, shaken for 2 hr. in a shaker incubator at 30oC, centrifuged and the supernatant used as testing sample. 2mg trypsin enzyme powder was dissolved in 50 ml of sodium phosphate buffer at pH 7.5. The reaction mixture involved 250 mg gelatin, 5 ml trypsin solution and 5 ml crude protein extract solution mixed to dissolve in a vortex mixer, incubated at 37oC for 2 h. and then cooled to 4oC. Samples containing trypsin inhibitors solidified in 15 minutes, while those without remained liquid indefinitely due to hydrolysis of gelatin by the added trypsin enzyme which was not inhibited. Control sample results for protein extracts from raw flour were used to confirm the results.
Detection of Haemagglutinins (Hgg)
Haemagglutinins were again detected according to Reddy et, al 1986 in the crude protein extracts [3]. The crude protein extract solution in 0.1N sodium acetate was precipitated by mixing in 1:1 ratio with 6 M sodium hydrogen sulphate solution, followed by separation by centrifugation. The precipitate was re-dissolved in 0.1N sodium acetate. 10 ml of this solution were mixed with 1 ml of blood from rabbit in saline solution. Precipitation and haemolysis of the red blood cells were checked as evidence of presence of haemagglutinins. Control sample results for protein extracts from raw bean flour were used to confirm the results.
Bean Samples |
60 min boiling |
15 min autoclaving at 121℃ |
||
Ti |
Hgg |
Ti |
Hgg |
|
All bean flours |
+ |
+ |
- |
- |
Bean protein extract |
+ |
+ |
- |
- |
Pigeon peas |
+ |
+ |
- |
- |
Ti = Trypsin inhibitor; Hgg = Haemagglutinins; + = Presence; - = Absence |
Table 2 shows the effect of roasting, drum drying, fine milling and boiling for 15 minutes and extrusion cooking at various temperatures on Ti and Hgg. Roasting inactivated Ti and Hgg completely, but created undesirable off-flavour and discoloration. Extrusion cooking inactivated Ti and Hgg at temperatures above 1000C, but left traces of Hgg which were eliminated only at 1400C. Both drum drying and fine milling together with boiling for 15 min were ineffective in elimination of the two nutritional stress factors.
Processing |
|
||||
Method |
Ti |
Hgg |
Off- flavour |
Discolouration |
|
Roasting |
- |
- |
+ + + |
+ + + |
|
Extrusion cooking (80Oc) |
+ |
+ |
- |
- |
|
Extrusion cooking (100Oc) |
- |
+ |
- |
- |
|
Extrusion cooking (140Oc) |
- |
- |
- |
- |
|
Drum drying |
+ |
+ |
- |
- |
|
Fine milling |
+ |
+ |
- |
- |
|
Ti = Trypsin inhibitor; Hgg = Haemagglutinins; - = Absent; + = Present; + = slightly present; + + + = Intensive development |
|||||
The effect of lactic fermentation of UNImix porridge on Trypsin Inhibitors (Ti) and haemagglutinins (Hgg) is shown in Table 3. The UNImix samples had their pH reduced to 3.0-3.2 and titratable acidities increased to between 0.8 – 1.0 % lactic acid equivalent on fermentation. There were no significant differences in these parameters between the samples. Fermentation alone without boiling did not affect either Ti or Hgg, while Hgg persisted in all fermented samples irrespective of cooking time. Trypsin inhibitors in UNImix containing soybeans and soybeans flakes were eliminated by a combination of fermentation and boiling for 3 and 5 minutes respectively. However, presence of trypsin inhibitors in fermented UNImix containing kidney beans persisted even after boiling for up to 15 minutes.
Cooking |
Soy flakes maize |
Soy bean maize |
Kidney bean maize |
|
|||||
(MSF) |
|||||||||
Ti |
Hgg |
Ti |
Hgg |
Ti |
Hgg |
||||
3 |
+ |
+ |
- |
+ |
+ |
+ |
|||
5 |
+ |
+ |
- |
+ |
+ |
+ |
|||
10 |
- |
+ |
- |
+ |
+ |
+ |
|||
15 |
- |
+ |
- |
+ |
+ |
+ |
|||
pH |
3.20 |
3.10 |
3.00 |
||||||
TA |
0.80 |
0.90 |
1.00 |
The presence of Hgg in UNImix porridge persisted irrespective of processing treatments with exception of extrusion-cooking and roasting, whether it contained kidney beans or soybeans. In particular, boiling of UNImix porridge containing kidney beans for up to 2 hours failed to eradicate its presence an indication of unlikelihood of its elimination in recipes containing kidney beans and cooked under normal households’ procedures [1]. This raises questions on harmfulness of Hgg on ingestion, given that no problems have been reported in communities consuming such diets. Reddy et al 1986 reported no problems associated with consumption of kidney beans diets cooked and processed differently but with detectable Hgg [3]. This fact can indirectly be collaborated by reported improvement of rehabilitative value and efficacy of High Energy porridge (HEP) containing kidney beans by lactic fermentation and presumably with residues of Hgg after cooking on clinically malnourished children in Uganda [11].
Current studies on replacement of extrusion-cooking with lactic fermentation using Uji starter culture showed that fermentation alone and even in combination with boiling of UNImix porridge containing kidney beans for up to 15 minutes was unable to eliminate Ti and Hgg, again demonstrating either stability of Ti and Hgg in kidney beans against inactivation under these conditions or their presence in such high quantities in Kidney beans requiring more time for inactivation at the given temperature. However, the Ti in the lactic fermented UNImix porridge containing soybeans but not Hgg was eliminated within 3-5 minutes, implying either more heat labile Ti in Soybeans than those in Kidney beans or their presence in such lower levels requiring shorter time for inactivation on basis of their inactivation kinetics. This finding however, justifies replacement of extrusion-cooking with lactic fermentation in production of UNI mix containing soybeans on the basis of elimination of Ti on minimal boiling for 3-5 minutes, considering that any residue Hgg are apparently harmless. Coincidentally lactic fermentation is reported to improve cook ability of porridges by enhancing starch gelatinization cycles during boiling thus lowering energy requirement unlike in extrusion-cooking, and making it appropriate for production of UNImix porridge at house hold level for nutritional intervention in children by mothers in Africa [7, 12-14].The fact that strains of Leuconostoc mesenteroides involved in lactic fermentation of Idli (an Indian legume-based product) have been reported to inactivate Hgg is a clear indication of diversity of LAB strains which can be harnessed and formulated in to desirable starter cultures for production of therapeutic porridge rations like UNImix porridge with desirable nutritional functionalities, which can be produced at household level in Africa for nutritional intervention and rehabilitation on sustainable basis instead of relying on industrially extruded-cooked porridge rations through donations [3, 10, 15-17].
- Mbugua SK, Keya EL. UNImix quality evaluation and development. A report for UNICEF Somalia Country Office, Nairobi. Department of Food Technology and Nutrition, University of Nairobi.
- Maritim G, Mason J. 1994. Report of a workshop on the improvement of the nutrition of refugees and displaced people in Africa. Machakos, Kenya, 5 – 7 December 1994. An initiative by UNACC Sub- Committee on Nutrition, UNHCR, WFP, Applied Human Nutrition (ANP) OF University of Nairobi and GTZ- Germany.
- Reddy NR, Pierson MD, Salunkhe DK. Legume based Fermented Foods. CRC Press Inc. U.S.A. 1986;155-170.
- Mbugua SK, Ledford RA and Steinkraus KH. Gaschromatographic determination of mono-, di- and trisaccharides in uji flour ingredients during fermentation. Chem-Microbiol Technol. Lebensm. 1983;8:40-45.
- Onyango C, Okoth MW, Mbugua SK. Effect of drying lactic fermented uji (an East African sour porridge) on some carboxylic acids. J Sci Food Agric. 2000;80(13):1854-1858.
- Barugahara Evyline Isingoma, Mbugua K Samuel, Karuri Edward, and Gakenia Wamuyu Maina. Socioeconomic and Demographic Factors Influencing Feeding Practices, Morbidity Status, and Dietary Intakes of Children Aged 7–24 Months in Rural Uganda. Ecology of Food and Nutrition. 2017;56(1):1-16.
- Mbugua SK. Nutritional and fermentation aspects of "Uji" produced from dry milled maize flour (Unga baridi) and wet milled whole maize. Chem. Mikrobiol. Technol. Lebensm. 10:154-161.
- Mbugua SK and Njenga J. The antimicrobial activity of fermented Uji. Ecology of Food and Nutrition. 1992;28(3): 191-198.
- Batista NN, Ramos CL, de Figueiredo Vilela L, Dias DR, Schwan RF. Fermentation of yam (Dioscorea spp. L.) by indigenous phytase-producing lactic acid bacteria strains. Braz J Microbiol. 2019;50(2):507-514. doi: 10.1007/s42770-019-00059-5
- Lorri WSM. Nutritional and microbiological evaluation of fermented cereal weaning foods. Ph.D. Thesis Dept of Food Science. Chalmers University of Technology Goteborg Sweden. 1993.
- Namayengo FM 2001. Substitution of High Energy Milk (HEM) with High Energy-High Protein Lactic Fermented kidney bean -maize porridge (HEP), in rehabilitation of clinically malnourished children at Mlango Hospital, Uganda. MSc Thesis Dept. Food Technology and Nutrition. University of Nairobi.
- Barugahara Evyline Isingoma, Mbugua K Samuel, Karuri G Edward and Gakenia W Maina. Performance of Nutritionally Optimized Millet Porridges in the Rehabilitation of Severely Malnourished Children at Mulango National Referral Hospital, Uganda. Journal of Advances in Medicine & Medical Research. 2016;18(2): 1-12.
- Mbugua SK. A method for manufacture of a fermented cereal product, African Research and Invention Property Office (ARIPO) Patent No. AP 122, 30.4.1991 and U.K Patent G.B 2.225 922B, 20.6.1992
- Mbugua SK, Ahrens RA, Kigutha HN, Subramanian V. Effect of Fermentation, malt flour treatment and drum drying on nutritional quality of Uji. Ecology of Food and Nutrition. 1992;28(4):271-277.
- Yao AA, Dortu C, Egounlety M, Pinto C, Edward VA, Huch M, et al. Production of freeze-dried Lactic Acid Bacteria starter cultures for cassava fermentation in to Gari. African Journal of Biotechnology. 2009;8(19):4996-5004.
- Holpzafel W. Use of starter cultures in fermentation on a household scale. Food Control. 1997:8(5-6):241-258.
- WHO,1996:Fermentation- Assessment and Research; Report of a joint FAO/WHO Workshop on fermentation as a household technology to improve safety, in collaboration with the Department of Health, Republic of South Africa Pretoria, South Africa, 11 – 15 December 1995. Food Safety Unit, Division of Food and Nutrition, WHO. 1996.