1. Introduction
Yoghurt is also known as a concentrated form of milk, made from selective bacterial fermentation [
1]. Over the past years, yoghurt has gained the attention of researchers around the globe. For instance, Greek yoghurt was widely known for its thin structure before the introduction of high-protein yoghurt due to its global marketing potentials. However, formulation of thick, creamy yoghurts have suppressed the market of Greek yoghurt [
2]. This is because scientists claim that thick and creamy yoghurts were said to be the primary option for consumers compared to the thin and watery type of yoghurt [
3]. The consumers’ stipulations for yoghurt will tend to increase in the future due to its potent beneficial effects and due to its inclusion of fewer additives [
4] and its nutritional value [
1]. Regardless of age group, yoghurt possesses the ability to increase amino acids content of a protein and may enhance the synthesize of proteins in the muscles [
5]. Yoghurt tends to contribute to faster satiety and is thereby advantageous for those in calorie-restricted diets [
6]. The bacterial strain in yoghurt makes it an ideal source of anti-obesity agents, and studies have proven that these bacterial cultures are able to tolerate the acidity in the gut and survive in the intestinal tract [
7]. Thus, lipolysis, amelioration of cognitive function, and reduction in the absorption of cholesterol are commonly documented features in those who consume yoghurts [
8]. Moreover, bacterial fermentation processes during yoghurt preparation transform the lactose in milk to lactic acids, which further gives the option for lactose-intolerant subjects to consume yoghurt without any allergic reactions [
9]. In these, integration of natural medicinal plants into yoghurt is a new insight in biomedical research.
Elateriospermum tapos (
E. tapos) is a monotypic plant, a tropical canopy found in the deep forest of Southeast Asia, commonly in Malaysia, Thailand, Sumatera, and Java [
10], and has raised our interest due to its beneficial composition.
E. tapos belongs to the family Euphorbiaceae and is known as “
buah perah” locally [
11]. Numerous bioactive compounds have been identified from
E. tapos extract, including flavonoids, tannins, alkaloids [
12], linolenic acids, polyunsaturated fats [
10], phenols, saponins, sterols, proteins, and iodine [
13].
E. tapos extract has been proven to enhance antioxidant activity, inhibit pancreatic lipase [
11], ameliorate stress hormone, and act as an anti-obesity agent [
10]. Due to its potential health effects,
E. tapos recently has attracted researchers’ attention to pursue further investigations in animal studies. According to the recent scientific findings, yoghurt is usually flavoured via natural plants or fruits to provide colour and bioactive compounds [
1]. As such, mixture of
E. tapos extraction, a local fruit, into yoghurt gives rise to a whole new formulation of yoghurt product. However, there is not any previous study that has introduced or evaluated the nutritional composition of
E. tapos yoghurt or assessed its toxicity. Hence, this article discusses the novel yoghurt formulation supplemented with ethanolic extraction of
E. tapos and its nutritional composition and toxicological evaluation on SD rats.
3. Discussion
This study is divided into two phases. In the first phase, also in vitro, the formulated
E. tapos yoghurt was analysed for its nutritional composition. According to the cut-off point for nutritional composition indicated by European Union (EU) regulations, the formulated
E. tapos yoghurt can be classified as a low-sugar and -protein yoghurt. This is due to its composition, which is <5 g sugar content per 100 g of
E. tapos yoghurt and 2.6 g of protein content per 100 g of
E. tapos yoghurt servings [
14]. According to EU guidelines, dairy products such as yoghurt must contain at least 2.7 g/100 g servings for total protein and a maximum of 5 g/100 g of servings for sugars, while for fat should be <3 g/100 g per serving. However, for fat content, the
E. tapos yoghurt shows a median threshold with 3.6 g per 100 g serving [
14]. It contains an appreciable amount of total dietary fibre at 1.2%, providing 9.6 kJ, which enhances satiety effects and reduces energy intake [
15]. It provides up to 33 kJ of calorie from digestible carbohydrates [
16].
In the second phase, the formulated E. tapos yoghurt was tested on female SD rats for safety analysis. The toxicity study was evaluated through acute and subacute toxicity studies (repeated dose). In acute toxicity study, AT 2000, at a single dose of 2000 mg/kg per body weight did not cause any mortality, behavioural abnormalities, postural changes, or drastic body weight changes. However, slight reduction in bodyweight was seen in ST 250 in the first week. Even so, the weight gain and calorie intake were comparable between the ST 250 and the control group during the rest of the experimental period. No weight loss was documented after the first week. Other groups in the subacute and acute study did not reveal any treatment dependent body weight changes or weekly food intake.
The kidney and liver are the most vital organs to rule for toxicity in both animals and humans. The gross weight measurement of kidney could indicate some sort of serious adverse effects. For instance, as noticed in this study (SST 1000 group), the increase in kidney mass could be due to hydronephrosis or nephrotoxicity, as reviewed by previous literature [
17]. However, the contraindication of liver profile test results, such as a decreased level of urea, creatinine, globulin, and ALP and AST in the acute or repeated-dose study, confirms the absence for the possibility of hydronephrosis or nephrotoxicity due to
E. tapos yoghurt’s consumption.
Meanwhile, the level of urea, ALP, AST, and creatinine are some of the biomarkers for renal functionality [
18]. Decreased levels of urea, globulin, and creatinine in serum correlates with severe malnutrition or liver diseases [
19], while significantly low levels of serum ALP and AST observed, as in subacute phase, contraindicate the possibility of developing malnutrition or liver disease. This is because low levels of ALP and AST prove the absence of protein malnutrition, as in the case of a low-protein diet, where the levels of ALP and AST will increase according to the severity [
20]. Moreover, a low-protein diet also is an indicator for decreased levels of urea in serum [
21,
22]. The primary reason for this is mainly because urea is the end metabolite of the protein catabolism process by the liver [
23], while creatinine is the product of creatinine metabolism in the muscles [
24].
In contrast, decreased levels of globulin together with increased levels of albumin are a simple indicator for the immune system maturation process. This is because maternally obtained antibodies tend to degrade within the first 6 weeks of birth, and during this period, albumin synthesize will increase [
25]. This is an indicator for normal liver formation, and since the SD rats used in this study were 6-week-olds, this hypothesis is acceptable. Non-significant changes observed in all other liver and kidney profile tests in both acute and subacute study may suggest that
E. tapos yoghurt does not have adverse effects on kidney and liver.
Haematological analysis in toxicity study shows the possibility of toxic effects induced by test material. From the blood biochemistry, a reduction in WBC, specifically lymphocytes, was recorded in both ST 500 and SST 1000 study, while low levels of eosinophils and MCV were documented in the SST 1000 and AT 2000 groups, respectively. Both lymphocytes and eosinophils are involved in immune defence, and usually a slight reduction in these is not something to be concerned with. However, significant reduction of lymphocytes and eosinophils in the animal model usually relates to chronic stress level, such as chronic renal failure or inflammation [
26]. However, normal architecture and absence of inflammation in histopathological evaluation of liver, kidney, and heart in both acute and subacute toxicity study confirms the absence of any form of injury to liver or kidney. Low levels of MCV are a clinical sign either in anaemic condition [
27] or psychological stress [
28]. Yet, normal levels of HB, RBC, MCHC, and MCH rule out the possibility of anaemic condition in treated rats. Thus, the slight decrease in lymphocytes, eosinophils, and MCV most probably could be incidental instead of due to a treatment-related effect. Non-significant changes observed in all other blood biochemistry parameters in both acute and subacute study may suggest
E. tapos yoghurt does not have effects on haematological products.
From the histopathological perspective, liver, kidney, and heart are the primary organs to be affected in case of stimulatory effect of toxins. Observation on gross morphology on kidney, liver, and heart did not show any form of abnormal texture, colour, or hypertrophy in both acute and subacute study in comparison to AC, SC, or SSC groups. Moreover, organ weight could be a marker of the pathological condition of the rats. Overall, there were no significant differences in ROW documented for liver, kidney, and heart in both acute and subacute studies in comparison with their respective control groups. Microscopic sectioning and staining of liver showed no evidence of inflammation or necrosis. Instead, a normal lobular arrangement was observed. There were neither manifestations of glomerulosclerosis nor scarring of parenchymal tissue in the renal tissue. In the heart, absence of myocardial necrosis was confirmed in both acute and repeated-dose study, further proving the safety of E. tapos yoghurt. In consideration with the data obtained from the nutritional analysis and toxicological evaluation study, it can be concluded that single-dose administration of up to 2000 mg/kg/day is a tolerable dosage.
4. Materials and Methods
4.1. Collection and Confirmation of Plant Species
The fresh E. tapos plant was obtained from Research Centre of Forest Research Institute of Malaysia (FRIM), Pahang. The plant materials were identified and deposited at the Medicinal Plant division of FRIM followed by the separation of E. tapos seed. The seed was then sent for confirmation at Herbarium Biodiversity Unit (voucher code: UPM SK 3154/17) at the Institute of Bioscience, Universiti Putra Malaysia.
4.2. Ethanol Extraction of E. tapos Seed
The
E. tapos seed was washed with running tap water to remove any external material. About 500 g of seed was soaked in 2000 mL of 95% ethanol in a 2 L conical flask. The conical flask was wrapped with aluminium foil and was left at room temperature for seven days. On the 7th day, the supernatant was collected and filtered through a Whatman paper N°1 filter paper. The solid residue was then repeatedly extracted 3 times with ethanol. The filtrates from each extraction was combined, and all the solvent was evaporated under reduced pressure using a rotary evaporator to yield the crude ethanolic extract of each
E. tapos seed [
29]. The obtained crude extracts were then mixed with maltodextrin in the ratio of 1:1 [
30]. The samples were stirred at room temperature and oven dried overnight at 45 °C. The final extracted powder was then stored at −20 °C until further usage.
4.3. Preparation of E. tapos Yoghurt
The formula for yoghurt preparation was adapted and modified from methods described elsewhere [
31]. About 100 mL of full cream (Dutch Lady Purefarm UHT) milk was pasteurized at 70–75 °C for 30 min using the microwave and cooled down to 45 °C at room temperature. The probiotic starter culture (0.06%
w/
w) consisting of a mixture culture of lactose, milk,
Streptococcus thermophilus APC151,
Lactobacillus delbrueckii subsp. Bulgaricus ATCC 11842, and autolysed yeast purchased from New England cheesemaking supply company was added to the cooled milk. This was then further incubated in the yoghurt maker (Pensonic PYM-700) at 40–45 °C for 7 h until the pH dropped to 4.5–4.6. The final product of the yoghurt was then refrigerated at 4 °C overnight. The following day, a stock
E. tapos yoghurt solution was prepared by dissolving 2 g of
E. tapos fleshes of powder into 100 mL of yoghurt [
31].
4.4. Color and Visual Appearance
A colorimeter (Chroma meter CR-400, Minolta, Osaka, Japan, equipped with illuminant/observer D65/2°) was used to study the colour changes in yoghurt during the fermentation process. The result was documented as lightness or darkness and yellowness or blueness, as described elsewhere [
32].
4.5. Determination of pH at 25 C
The pH values of the formulated yogurt were measured using digital potentiometer (420 Benchtop, Orion Research Inc., Beverly, MA, USA) [
1].
4.6. Titrable Acidity of Lactic Acid
The titrable acidity of lactic acid was determined according to the method described in AOAC (2000). In this, 0.1 M of NaoH was titrated into 10 mL of yoghurt [
33]. The acidity was then expressed as gram of lactic acid/100 g using the formula below:
4.7. Determination of Total Sugar
The refractometric method was used to determine the total sugar content in the yoghurt. In this, about 2 to 3 drops of yoghurt was pipetted into the main prism. The appearance of boundary lines in the refraction field was recorded, and the refractive index was identified [
34].
4.8. Determination of Fat
Gerber protocol was adapted to determine the fat content. In these, 10 mL of ammonia was added to the 100 mL yoghurt and mixed well. Simultaneously, 10 mL of 1.825 g/L sulfuric acid (H
2SO
4) was pipetted into the butyrometer. The mixture of yoghurt was then gently pipetted into the butyrometer, allowing the formation of a thin layer on top of the H
2SO
4 acid. Then, 1 mL of 0.815 g/L amyl alcohol was dispensed into the butyrometer, inverted, and was centrifuged for 5 min [
35]. After centrifuging, the fat content was determined using the formula below:
A = Reading obtained at the base of fat column;
B = Reading obtained at the crest of the fat column.
4.9. Determination of Protein Content
The Kjeldahl method was used to measure the protein content. Formulated yoghurt was placed into Kjeldahl flask and digested with H
2SO
4 and catalyst. This was followed by dilution with H
2O and neutralization with sodium thiosulfate to obtain boric acid solution. Hydrochloric acid was then used to titrate the borate anions, which eventually forms nitrogen [
36]. The crude protein was calculated by multiplying with the conversion factor of 6.38 using the formula below:
4.10. Determination of Carbohydrate
Phenol sulfuric acid method was used to determine the carbohydrate content. First, 200 µL of 5% of phenol was added to the 200 µL of yoghurt in a test tube. This was followed by adding 1 mL of concentrated H
2SO
4 and vortex. The mixture in the test tube was then incubated in room temperature for 60 min and was read using a spectrophotometer at 490 nm absorbance [
37].
4.11. Determination of Energy
The energy content of the formulated yoghurt was calculated based on the method JKM F 1205 based on the guidance of Nutrition Labelling and Claims by Food safety and quality Division MOH described in the Malaysian food composition database [
38]. The formula used for energy (kcal) calculations are as below:
4.12. Determination of Total Dietary Fibre
Dietary fibre content was determined according to the procedure elsewhere [
33]. In this, the formulated yoghurt was freeze dried until the powder form was obtained. The powder form of yoghurt was then placed into digestion flask, and 200 mL of 1.25% of H
2SO
4 was added. It was boiled for about 30 min with frequent rotation until the samples became completely wet. Thereafter, the samples were filtered with fluted funnel and washed with boiling water. The residue was again washed with 200 mL of 1.25% of H
2SO
4 and boiled for 30 min via reflux condenser. Thereafter, the samples were filtered with an asbestos mat, and the residue was again washed with boiling water, followed by 10 mL of alcohol, and dried at 110 °C (W
1). The residue was then transferred into the muffle furnace at a controlled temperature of 525 to 550 °C, and the ash material was used to obtain the value of W
2. The loss of weight represents the crude fibre [
33].
4.13. Survival of Lactic Acid Bacteria
The method was adapted and modified from Vinderola et al., 2003. In this, approximately 25 mL of yoghurt was diluted with 225 mL of peptone H
2O in a bag mixer 400 (Interscience, St. Nom, France). The dilution was then spread on a Petri dish containing Rogosa agar and incubated at 37 °C in anaerobic jars for 72 h. After 72 h, the colony formed was counted and expressed as colony-forming units per gram (CFU/g) [
39].
4.14. Microbiological Analysis
Survival of Lactobacillus species in the yoghurt was measured on day 1 and 90 according to the method described by Tontul et al., 2018. In this method, 1 g of yoghurt was diluted with 9 mL of Ringer solution (Merck KGaA, Darmstadt, Germany) and vortexed, and further dilutions were made. The dilutions were then spread in a Petri dish containing Rogosa agar. The colony formed was counted, adjusted to pH 6.5 by adding on 0.1 M of NaOH, and incubated at 37 °C in anaerobic jars for 72 h. For
Streptococcus thermphillus, the colony was counted by spreading the dilutions in a Petri dish containing M17 agar (Merck KGaA, Darmstadt, Germany), which was incubated aerobically at 37 °C for 48 h [
32].
4.15. Experimental Animals
All animal-related procedures were conducted under the approval of the Animal Care and Use Committee of the Management and Science University (MSU). The animal ethics committee of MSU granted approval for this study under the animal ethics number of AE-MSU-073. Young adult female Sprague Dawley (SD) rats weighing between 150 g and 200 g (6 weeks old) were used to access acute and subacute toxic effects of
E. tapos yoghurt for the in vivo study. All rats were acclimatized for 1 week in a temperature-controlled room (22 ± 3 °C) at 12/12 h light/dark cycle. The rats were fed with standard rat pellets containing 306.2 kcal/100 g with 48.8% carbohydrate, 21% protein, and 3% fat, which is equivalent to 12.8 kJ/g, with ad libitum availability. Body weight and 24-hour food intake (kJ) were measured weekly [
40].
4.16. Acute Toxicity Study
The acute toxicity study was performed according to the protocol described in OECD 425 guidelines. Rats were divided into 2 groups (n = 8), where the control group received normal saline (bottle-feeding), while the treatment group received 2000 mg/kg of E. tapos yoghurt (force-feeding) for 2 weeks. All rats were observed every 4 h for the next 14 days for any abnormalities in behaviour pattern until euthanasia period. At the end of experiment, all rats were euthanized with CO2. Blood was collected in EDTA and plain tube for further biochemical and haematological analysis. Liver, kidney, and heart were excised, weighed, observed macroscopically, and preserved in 10% neutral buffered formalin at room temperature.
4.17. Subacute Toxicity Study (Repeated Dose)
Subacute toxicity test was performed according to OECD 407 guidelines on repeated dose of 28-day oral toxicity study in rodents. Rats were divided into 6 groups (n = 8), and E. tapos yoghurt was orally administered (force-feeding) once daily at a dose of 250, 500, and 1000 mg/kg, while the subacute control (SC) and satellite control (SSC) received a normal saline (bottle-feeding). All rats were observed every 4 h for any abnormalities in behaviour patterns until the euthanasia period. At the end of experiment, all rats were euthanized with CO2. Blood was collected in EDTA and plain tube for further biochemical and haematological analysis. Liver, kidney, and heart were excised, weighed, observed macroscopically, and preserved in 10% neutral buffered formalin at room temperature.
4.18. Full Blood Count and Plasma Biochemistry
Serum was used to access plasma biochemistry, such as haemoglobin (HB), red blood cell (RBC), red cell distribution width (RDW), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet, and white blood cell (WBC), using automated BC-2800 VET/Mindray machine. Liver profile, such as level creatinine, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine aminotransferase (ALT), sodium, potassium, chloride, urea, creatinine, and kidney profile test (total protein, albumin, globulin, albumin-globulin ratio), were measured using Alere Cholestech LDX® Analyzer (Alere, UK).
4.19. Histopathological Analysis
Tissue processing was performed on all the preserved organs (kidney, heart, and liver). Sectioning was performed to obtain a thin layer of paraffin ribbon with tissue thickness between 4 μm to 7 μm followed by ribbon fishing. Paraffin ribbon was placed in the water bath at a temperature range of 40 °C to 45 °C and was transferred onto a glass slide. All slides were stained using haematoxylin and eosin (H&E) stain. Tissues were then observed under the light microscope, and histological changes were captured [
13]. The presence of lesions was scored according to the guideline provided elsewhere [
13]. All the scoring was validated by two independent certified pathologists from UPM.