Cookies rich in iron (Fe), folic acid, cobalamin (vitamin B12), and antioxidants: a novel functional food potential for adolescent with anemia

Editorial note

Editorial Note (20^th October 2023): The F1000 Editorial Team has not yet received a new version of this article, as detailed in the Editorial Notes published on 16^th June and 4^th August 2023, despite repeated chasing. The F1000 Editorial Team will no longer be requesting a new version from the authors. Readers should be aware that there is key information missing from the article regarding the methodology and analysis of this research; this information had been previously given to and verified by the F1000 Editorial Team, but the authors have not yet updated their article. Peer review activity remains suspended until the authors publish a new version of this article.

Editorial Note (4^th August 2023): The F1000 Editorial Team has not yet received a new version of this article, as detailed in the Editorial Note published on 16^th June 2023. The F1000 Editorial Team is actively contacting the authors to request the new version of the article. Peer review activity remains suspended until the authors publish a new version of this article.

Editorial Note (16^th June 2023): Since publication, it has been brought to the attention of the Editorial Team that the article was missing key information regarding the methodology and analysis of this research. The Editorial Team requested further detail from the authors in March 2023. The authors provided an adequate response and were requested by the Editorial Team to create a new version of the article to include the additional details. Peer review activity has been suspended until the authors publish a new version of this article.

Introduction

Anemia is a serious public health problem faced by all countries in the world. According to the World Health Organization (WHO), 42% of children <5 years, 33% of women of productive age, and 40% of pregnant women in the world have anemia. In Indonesia, Basic Health Research (Riskesdas) shows an increase in the prevalence of anemia in adolescents aged 15-24 years. In 2007, the prevalence of adolescent anemia in Indonesia was only 6.90% (Riskesdas, 2007), which then increased to 18.40% in 2013 (Riskesdas, 2013) and 32% in 2018 (Riskesdas, 2018). The impact of anemia is complex, ranging from immunity (Hassan et al., 2016) to work productivity (Blakstad et al., 2020), growth (Soliman et al., 2009) to intelligence (Bahrami et al., 2020), and physical fitness (Tsai et al., 2019) to maternal, fetal and child health (Georgieff, 2020).

Various interventions need to be done to speed up the treatment of anemia. In recent years, anemia prevention and treatment research among children and adolescents in Indonesia has been largely relying on nutritional education (Zuraida et al., 2020; Sari et al., 2018). Additionally, The government of Indonesia has its official program, namely the provision of iron and folic acid supplementation in the form of Blood Added Tablets (TTD) in school-aged teenagers. Although the program's scope is quite high, which is 76.2%, its effectiveness is relatively low because only 2.13% consume TTD as recommended ≥52 items per year (Riskesdas, 2018). Moreover, a study by Apriningsih et al. (2020) stated that highschool female’s adherence to consume iron-folic acid tablets in Depok was relatively low (45.6%). Other similar study found that adolescent girls who consumed at least one tablet per week was only 9% and 18% in East Nusa Tenggara and East Java, respectively (Alfiah et al., 2020). Therefore, alternative efforts such as food fortification become important to do, especially in snacks that are mostly preferred by teenagers. In addition to promising results in terms of decreasing the problem of micronutrient deficiency (Dewi & Mahmudiono, 2021), food-to-food fortification in snacks is also potentially effective because adolescents aged 18-25 years tend to consume snacks ≥3 times per day which contributes to 25% recommended dietary allowance (RDA) or average daily dietary intake level (USDA, 2012).

Many local Indonesian foodstuffs are of high nutritional value (Permatasari et al., 2021), for example, porang tubers. Porang's tubers (Amorphophallus oncophyllus) can be used as potential glucomannan source flour in Indonesia (especially the original regency, Madiun, East Java, Indonesia) because of the high glucomannan content (Harmayani et al., 2014). The bulbs are cultivated in Indonesia as secondary plants under teak, mahogany, and sonokeling plantations (Yanuriati et al., 2017). As a source of glucomannan, people have many benefits as functional foodstuffs (Harmayani et al., 2014; Yanuriati et al., 2017). Glucomannan properties can bind to other components such as proteins, minerals, and active compounds such as antioxidants that form a matrix of tissues in the food developed from it (Herlina et al., 2015). Glucomannan has been widely used in the food industry as a thickener, gel-forming, texture, water binder, and emulsion stabilizer, including in cookie making (Imeson, 2009; Mikkonen et al., 2009; Zhang et al., 2005). However, apart from having a high glucomannan content, Wardani and Arifiyana (2020) found that porang tubers also contain high levels of calcium oxalate (2.6030% w/w) that should be considered since it can cause itchy mouth, irritation, and trigger the development of kidney stones (Ulfa & Nafi’ah, 2018).

In addition to porang's tubers, another local foodstuff that is also known to have many health benefits is moringa leaves. Moringa oleifera is known as an excellent food source that is easy to digest and rich in protein (Fahey, 2005). According to Sultana and Anwar (2008), moringa leaves have many valuable compounds such as protein, vitamins, calcium, iron, folic acid, and antioxidants (carotenoids, flavonoids, and phenols) (Sultana & Anwar, 2008). Various developing countries in the world feed their children with moringa (Kasolo et al., 2010). Busani et al. (2011) reported that the presence of various minerals and vitamins helps boost immunity against various diseases, especially moringa leaves contain various amino acids (Busani et al., 2011). In addition, the levels of iron, folic acid, and antioxidants contained in it also have the potential to become an anti-anemia agent (Mo et al., 2013; Kurtoglu et al., 2003). On the other hand, moringa oleifera also contains 5% saponins and 3.1% phytate, which are considered as anti-nutritional factors (Makkar et al., 1996).

Indonesia's third potential foodstuff, which has been known globally, is tempe. Tempe is made from soybeans fermented by Rhizopus spp mushrooms such as R. oligosporus, R. stolonifer, and R. oryzae with several characteristics such as white, compact texture, and mixed distinctive flavors of mushrooms and soy flavors (Astuti et al., 2013). Black soybean tempe has potential functional characteristics, as black soybeans contain phenolics, tannins, anthocyanins, cobalamin (B12), and isoflavones, as well as a higher antioxidant activity than yellow soybeans (Xu dan Chang, 2007). According to Nurrahman et al. (2011) black soybean tempe has the same level of fondness as yellow soybean tempe, and its antioxidant activity is higher (Nurrahman et al., 2011). In addition, Vitamin B12 (Cobalamin) in the tempe has the potential in the formation of red blood cells and its antioxidants to overcome red blood cell damage (Mo et al., 2013; Kurtoglu et al., 2003).

Therefore, the combination of porang’s tubers, moringa leaves, and tempe made from black soybeans can be used as processed products such as functional cookies as a snack for anemia adolescents. This study utilizes and formulates all three ingredients into functional cookies and determines levels of iron (Fe), folic acid, cobalamin (vitamin B12), and antioxidants.

Methods

This study is experimental with a completely randomized trial design (CRD) with three treatments and triplicates, and only moringa leaf flour and tempe flour (black-soybean) ingredients are differentiating treatments with a percentage increase in each formulation (see Table 1), the rest are the same or controlled. Ingredients or materials including porang flour, moringa leaf flour, tempe flour (black-soybean), stevia, skimmed milk, egg yolk, baking powder and margarine are obtained or purchased from the market in Yogyakarta, Indonesia and are in accordance with the Indonesian National Standard (SNI).

Results

Discussion

Anemia is a severe public health problem faced by all countries globally, and various interventions need to be done to speed up the treatment of anemia. One of them is utilizing local natural ingredients as functional food sources of iron (Fe), folic acid, cobalamin (vitamin B12), and antioxidants for adolescent anemia. In this study, researchers utilized and formulated all three local natural ingredients (a combination of people's tubers, moringa leaves, and black soy-based tempe) into functional cookies and determining levels of iron (Fe), folic acid, cobalamin (vitamin B12), and antioxidants. The selection of processing into cookie products is wrong as an alternative effort instant snack food that is majority preferred by teenagers without having to process it again (e.g., iron source chicken liver but must go through the cooking process, etc.). This research is a basic study, whose certainty of clinical benefits needs to be done further through pre-clinical studies and clinical trials in humans. However, the results of this study (cookie formulation) can be used as a reference in the dosing of preclinical trials and future clinical trials. The results of the ash and water content tests of these three cookie formulations showed a value in accordance with the Indonesian National Standard for cookie products (SNI 01-2973-1992), which is a maximum ash content of 1.5% and water content of no more than 5%.

Formulation 3 (F3) is a high iron cookie, amounting to 29.41 ± 0.65 mg/100 g, higher than F1 and F2 significantly (p < 0.05) (Table 4; Figure 1). In the scheme (Figure 5), iron is needed by the body for erythropoiesis or the process of red blood cell synthesis, precisely as the constituent material of hemoglobin (Singh, 2018). F3 cookies in this study have higher iron levels than the functional cookie of previous research 2020 which only has a level of 2.51 mg/100 g (Rahmat et al., 2020). In line with AKG (Angka Kecukupan Gizi) Indonesia in 2019, iron adequacy rates for ages 10-18 years in men range from 8-11 mg, in women 8-15 mg per person per day. Seeing this, F3 cookies are potentially a food source of iron, especially for adolescents with anemia.

Figure 1. F3 cookie has the highest Fe content.

In formulation 3 (F3) based on multivariate results, iron (Fe) levels were 29.41 ± 0.65 mg/100 g, significantly higher than F1 and F2 (p < 0.05).

In addition to iron height, F3 cookies have a fairly high antioxidant content of 24.41 ± 1.31%, although there is no significant difference with F2 cookies (Table 4; Figure 4). In addition to needing iron food sources to avoid anemia is also needed foods that have antioxidant abilities to protect red blood cell damage due to the occurrence of oxidative stress (Reactive Oxygen Species) (Lobo et al., 2010; Gwozdzinski et al., 2021).

Folic acid or vitamin B9 and cobalt (vitamin B12) also have an important role in minimizing the incidence of anemia. Folic acid collaborates with vitamin B12 in making red blood cells and helps iron function properly in the body (Figure 5) (Mahmood, 2014). Vitamin B9 works with vitamins B6 and B12 and other nutrients in controlling the amino acid homocysteine levels in increasing the effectiveness of red blood cell formation (Figure 5) (Mahmood, 2014). Formulation 3 (F3) is the best formulation when viewed from iron (Fe), folic acid (vitamin B9), cobalamin (vitamin B12), and antioxidant activity. F3 cookies have the highest levels of folic acid and cobalamine (308.24 ± 1.30 mcg/100 g and 109.76 ± 1.74 mcg/100 g) compared to F1 and F2 (Table 4, Figures 2 and 3). Cookie F3 has folic acid that is closest to AKG (Angka Kecukupan Gizi), which recommends 400 mcg per person per day (for men and women aged 10-18 years).

Figure 2. F3 cookie has the highest folic acid content.

In formulation 3 (F3) based on multivariate results, folic acid (vitamin B9) or folate levels were 308.24 ± 1.30 mcg/100 g, significantly higher than F1 and F2 (p < 0.05).

Figure 3. F3 cookie has the highest cobalamin content.

In formulation 3 (F3) based on multivariate results, the levels of vitamin B12 (cobalamin) owned amounted to 109.76 ± 1.74 mcg/100 g, higher than F1 and F2 significantly (p < 0.05).

Figure 4. F3 Cookie has the highest antioxidant activity.

Antioxidant activity is highest in F3 with a percentage of 24.41 ± 1.31. However, there is no meaningful difference (p > 0.05) between F2 and F3 (p = 0.355); which means that formulations F2 and F3 have the same antioxidant effect properties. In addition, F3 is the recommended formulation for in vivo tests on experimental animals to see further antio-oxidative effects.

Figure 5. Possible-mechanism of the Potential F3 cookie scheme as anti-anaemic.

Cookie innovation made from a combination of porang's tubers, moringa leaves, and tempe made from black soybeans has potential to be made a functional anti-anemia food or to minimize anemia comprehensively (see Figure 5). In addition, the sugar or sweetener used in this formulation is stevia which is clinically safer (not hyperglycemia) (Ashwell, 2015). However, further studies are needed in both experimental animals and humans to find out more about the clinical benefits.

Conclusion

The combination of porang's tubers, moringa leaves and tempe made from black soybeans, especially F3 has the potential to be used as a functional cookie processed product source of iron (Fe), folic acid, cobalamin, and antioxidants for adolescent anemia. As a novel functional food potential for adolescent with anemia, preclinical trials or in vivo trials on undernourished animal models and clinical trials on humans are urgently needed with reference to this Cookie F3.