Impact of Dehydrated Anchovy Powder on the Growth and Nutritional Well-Being of the Human Undernourished Population: Assessing Bioavailability and Nutritional Effectiveness through In Vivo Experimental Models

The research focuses on assessing the efficacy of dried anchovy powder as a dietary supplement, examining its bioavailability and impact on growth and nutritional status. The study, centered on Wistar male rats, acknowledges anchovies for their rich essential nutrients and potential health benefits. Employing a meticulously controlled experimental design, the research exposes experimental animals to various dietary interventions by integrating dried anchovy powder. This study investigates the availability of vital nutrients like proteins, omega-3 fatty acids, vitamins, and minerals in the dried anchovy powder to Wistar male rats. The effects of dried anchovy powder on growth parameters, encompassing body weight, length, organ development, and the nutritional status of the rats, are explored. Examining hematological and biochemical markers provide insights into the overall health of the experimental subjects. Additionally, the research delves into potential mechanisms underlying the observed effects, including nutrient absorption and metabolism. The outcomes of this study offer valuable insights into the potential of dried anchovy powder as a nutritional supplement and its role in enhancing the nutritional status of the malnourished people of India. These findings may have a direct impact on dietary interventions aimed at improving human nutrition and health.

Keywords

dried anchovy powder - malnutrition - growth parameters - animal feeding - wistar rat

Introduction

Malnutrition presents a multifaceted challenge in India, marked by insufficient access to a well-rounded and nourishing diet. The nutritional concerns in India, within the context of fisheries management, are intricately linked to the sustainability of fish as a valuable source of nutrients.[1] The depletion of fish stocks due to overexploitation has the potential to compound malnutrition by restricting access to essential nutrients. Therefore, implementing effective fisheries management strategies that prioritize sustainability is paramount to preserving ecological equilibrium and supporting the livelihoods of fishing communities.[2] Effectively addressing malnutrition entails advocating for consuming diverse fish species, considering socioeconomic implications, and actively involving local populations in sustainable fishing practices.[3]

Recent research reflects a growing interest in exploring natural sources for nutritional supplements, driven by the increasing recognition of their potential health benefits.[4] Anchovies are renowned for being a rich source of protein, with higher levels than many other animal protein sources. Additionally, fish contains almost essential amino acids, polyunsaturated fatty acids, vitamins and minerals, making it a valuable dietary choice. Specifically, as Kari et al[5] emphasize, dried anchovy powder has garnered attention due to its abundant essential nutrient content. As small pelagic oily fish found in trophic and temperate waters, anchovies are scientifically significant owing to their nutritional value and associated health advantages. These encompass pivotal roles in cardiovascular health, inflammatory disease prevention, infant neuronal development, fat glycemic control, and other health aspects.[6] Anchovies, classified in the Clupeiformes order of the family Engraulidae, are distributed in the Indian and Pacific Oceans, primarily in shallow coastal waters.[7] They vary in size, generally 8 to 32 cm in length, with distinct coloration.

According to the 2017 Food and Agriculture Organization report, anchovies and sardines constitute around 52% of global landings for small pelagic fish. In Malaysia, anchovy landings were reported at 28,894 tons in 2019, with Kedah contributing significantly. The role of anchovies remains prominent within Indian pelagic fisheries resources. In the year 2003, the respective contributions of anchovies to the total marine fish landings were 6%. Indian waters are home to 28 recorded species of anchovies, with significant contributions attributed to the Stolephorus, Engraulis, Thryssa, Setipinna, and Coilia genera. The anchovy fishery in India focuses on the coastal states of Andhra Pradesh, Tamil Nadu, and Kerala, collectively making significant contributions to the national anchovy catch.[8]

Recognized as an excellent protein source, anchovies offer a complete amino acid profile crucial for bodily functions. Their highly bioavailable protein ensures efficient absorption and contributes omega-3 fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), known for cardiovascular and cognitive health benefits. With essential vitamins and minerals, anchovies enhance their nutritional value, making them a versatile and health-promoting addition to diets.[9] [10] Despite their ecological significance and nutritional richness, anchovies may be labeled as “trash fish” due to factors like small size or perceived economic value. Since anchovies are consumed whole, they are considered as a source of calcium and phosphorus. However, recognizing their role in marine ecosystems and promoting sustainable harvesting challenges this perception.[11] Efforts to transform anchovies into a valuable and eco-friendly protein source involve identifying suitable species, ensuring sustainable harvesting, and developing efficient processing techniques.[12] Anchovies have significant potential in Indian fisheries, particularly in the Arabian Sea and the Bay of Bengal, addressing nutritional deficiencies with their protein and nutrient richness. Sustainable harvesting practices and efforts to promote their consumption could enhance their economic and healthy contributions to the Indian fisheries sector.[13]

This study aims to evaluate dried anchovy powder's nutritional effectiveness and bioavailability as a dietary supplement. The drying process is essential for anchovy preservation, extending its shelf life, concentrating nutrients, and promoting economic sustainability in fishing communities. Recognizing the pivotal role of anchovies in fisheries management is crucial for fostering a diverse fisheries industry, addressing protein deficiencies, and creating economic opportunities. Utilizing Wistar male rats, the research investigates the impact of dried anchovy powder on growth parameters, health markers, and underlying mechanisms such as nutrient absorption and metabolism. The findings carry implications for the development of functional food products or dietary interventions that can contribute to human well-being in the context of nutrition and health.

Materials and Methods

Production of Anchovy-Dried Fish Powder Employing Oven Drying

The manufacturing process of dried anchovy powder began with the acquisition of fresh anchovies from the local market, the species being identified as Stolephorus commersonnii based on the Food and Agriculture Organization species identification sheet. These anchovies were meticulously cleaned to eliminate sand particles and dirt before being arranged on a metallic tray. Subsequently, the anchovies underwent a 3-day drying period in an electric drier set at 50°C. Once dried, they were finely ground into a powder using a pulverizer equipped with a 250 μm mesh sieve (Model 160B; Jacobson Machinery Works, Minneapolis, Minnesota, United States). The resulting fish powder was then carefully packed in polypropylene bags and stored at −18°C until used. The detailed process flow chart for the production of dried anchovy powder is illustrated in [Fig. 1].

Fig. 1 Flowchart outlining the steps involved in producing dried anchovy powder.

Determination of Proximate Composition

The samples were analyzed for the proximate composition. The moisture, protein, fat, and ash content were analyzed using the validated method.[14]

Moisture

The moisture content was determined by drying a 10 g sample at 105°C in a thermostatically controlled hot air oven. The samples were taken in a preweighed petri dish and kept in an oven; the weight reduction was checked by repeatedly weighing and then cooling the sample in desiccators till the weight became constant. Moisture content was expressed in percentage.

W1= Weight of dry petri plate
W2= Weight of the sample in the petri plate
W3= Weight of the sample in the petri plate after drying

Crude Protein

Crude protein content was determined by the Kjeldahl nitrogen method, which involves digestion, distillation, and titration. About 100 mg of the sample was taken into a Kjeldahl digestion flask of 100 mL capacity. A few glass beads, a pinch of digestion mixture (CuSO4, K₂SO₄), and 10 mL of sulfuric acid were added. This was then subjected to digestion over a burner till the solution turned colorless. The digested and cooled solution was made up to 100 mL by adding distilled water. Ten milliliters of the made-up solution was then added to the reaction chamber of the micro-Kjeldahl distillation apparatus and rinsed down with distilled water. Two drops of phenolphthalein indicator and 40% NaOH were added till the indicator changed to pink. Distillation was performed for 4 minutes, and liberated ammonia was collected in 2% boric acid (10 mL) containing a drop of Tashiro's indicator. The amount of ammonia was determined by titrating with N/100 sulfuric acid until the solution turned pink.

x = Titer value of the sample
V = Total volume of digest
V1 = Volume of digest taken for distillation
W = Weight of sample taken

Crude Fat

Crude fat content was determined by the Soxhlet method. One gram of sample was weighed into a thimble and placed in a Soxhlet apparatus. The sample was extracted using petroleum ether for 3 hours at 100°C. After extraction, the solvent was evaporated at 80 to 100°C, and the fat content was weighed after cooling it in a desiccator.

W1 = Weight of the sample
W2 = Weight of the Soxhlet beaker
W3 = Weight of fat and beaker

Ash

To determine the ash content, silica crucibles were cleaned and kept in a muffle furnace at 600°C for an hour. This was then cooled in a desiccator, and the weight of the empty crucible (W1) was noted down. Sample (2 g) was taken in a crucible and was subjected to heating until the material got charred. The charred material was then transferred to a muffle furnace, previously set at a temperature of 650°C and heated to 6 to 8 hours until the material became white or grayish-white ash. The crucibles were cooled in a desiccator, and weight was noted (W3).

W1 = Weight of crucible
W2 = Weight of sample
W3 = Weight of ash in the crucible

Determination of Fatty Acid Profile

The analysis of fatty acids involves three steps: lipid extraction, preparation of fatty acid derivatives, and gas chromatographic (GC) analysis. The crude lipids in the gutted fish samples were extracted using a chloroform-methanol mixture.[15] Saponification of fats liberates fatty acids from triglycerides. The fatty acids are derivatized into their corresponding fatty acid methyl esters by reflexing with BF₃ methanol reagent,[16] and the fatty acid profile is analyzed using the gas liquid chromatography—Flame Ionization Detector (FID) method.

The GC is set at the required temperature with the optimum flow rate of nitrogen gas (N₂), the carrier gas in the chromatograph. Program of GC Injector 260°C; FID—275°C; capillary column, PE Elite 225 (30 m, 0.25 mm 1.d, 0.25 um) Carrier gas- Nitrogen at 0.6m/min; Air 30ml/min and Hydrogen 30 mL/min for FID temperature program-110°C. After an initial hold of 4 minutes, the temperature is programmed to rise from 2.7°C/min to 240°C and maintained at that temperature for 5 minutes; the split flow is 12 mL. Samples are identified by retention time by comparing with respective standards using the software. The area of each component is obtained from the computer-generated data, and concentration is calculated using the software by an external standard method. The fatty acid composition samples are expressed as g/100 g of total fatty acid content.

In Vivo Studies of Anchovy Dried Fish Powder in Male Wistar Rats

Preparation of the Feed

The feed was prepared for the experimental control animals by incorporating the anchovy dried fish powder (1.5%) into the standard diet. Positive control feed was prepared by incorporating casein (1.5%) into the standard diet, and the negative control was given a regular diet without any alterations.[17] The standard feed was powdered to maintain consistency, and the required ingredients were added, pelletized, and dried to attain a consistent shape and size ([Fig. 2]). Feed and water are provided ad libitum, ensuring that animals have constant access to both.

Fig. 2 (A) Control feed, (B) casein incorporated feed, and (C) anchovy dried fish powder incorporated feed.

Animal Feeding Trials

The experimental rats were handled following the guidelines for the regulation of scientific experiments on animals set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. These procedures were approved by the Institutional Animal Ethics Committee of ICAR-Central Institute of Fisheries Technology, Cochin, with approval no: CIFT/B&N/IAEC/2020–1(6).

The initial trials assessed the efficacy of various feeds in male Wistar rats, considering parameters such as feed intake, average weight gain, feed conversion ratio (FCR), and specific growth rate (SGR). Thirty-three Wistar albino male rats weighing between 190 and 210 g were chosen for the feeding trials. The rats were initially given a basal diet for 1 week to facilitate adaptation. Following this period, the rats were divided into three groups, each containing 11 rats. The first group/ negative control continued with the basal diet (control feed), the second group/positive control group received casein-incorporated feed, and the third group/experimental group received anchovy dried fish powder incorporated feed. Throughout the study, all rats had unrestricted access to both food and water. They were paired and housed in individual cages, adhering to a 12-hour dark/light cycle. Weekly recordings were made of the rats' body weights and measurements of their daily food and water intake.[18]

After the experiment, rats were euthanized in a CO₂ chamber maintained at a flow rate of 3 mL/min for 5 to 10 minutes, and tissues were collected after rinsing with an ice-cold saline solution. A portion of the liver tissue was preserved in a 10% buffered formalin solution for subsequent histopathology analysis. Blood samples were obtained from animal models without anticoagulants through cardiac puncture. The serum, separated through centrifugation, was then employed for hematological parameters.

Assessment of Growth Parameters in Experimental Animals

Feed Conversion Ratio

In animal studies, the FCR is a parameter used to measure the efficiency of converting feed into body weight gain. It indicated how much feed was required to produce an inevitable weight gain in the animals. The FCR was calculated using the following formula:

A lower FCR value indicates better feed efficiency, meaning less feed was required for a specific weight gain. Conversely, a higher FCR value suggested poorer feed efficiency, indicating that more feed was needed to gain the same weight.[19]

Specific Growth Rate

The SGR is a parameter used to measure animal growth rate over a specific period. It indicates how quickly the animals are gaining weight. The SGR is calculated using the following formula:

The SGR indicated the relative growth rate of the animals over the study period. A higher SGR value indicated a faster growth rate, while a lower SGR value suggested a slower growth rate. SGR can be useful for comparing the growth performance of different treatments or groups in an animal study.[20]

Blood Profiling

Blood serum parameters like lipid profile, liver enzymes, and renal function tests were analyzed by using an automated analyzer FUJI DRI-CHEM- NX 500.[21]

Histopathological Evaluation of Liver Tissue

The liver tissue was fixed in a 10% buffered formalin fixative overnight. Hematoxylin and eosin (H and E) staining was performed on sections of the liver samples that were 5 μm thick and placed in paraffin wax. The stained slides of liver tissues were observed using a light microscope fitted with a digital camera.[22]

Statistics

A two-way analysis of variance was performed, the significance of the hypothesis was tested at a 5% significance level (p < 0.05), and means were compared using Tukey's test. Statistical analysis was performed using SAS 9.3.

Results and Discussions

Dehydrated Anchovy Powder

Traditionally, anchovies were converted into a dried form due to their vulnerability to deterioration after death. The conventional process involves cleaning the fish, stacking them one upon another, and exposing them to sunlight for drying.[23] Value addition processes such as filleting, drying, and flavoring enhance the market appeal of anchovies by addressing challenges like their small size, seasonal availability, and perishability.[24] By creating diverse products with extended shelf life and improved flavor profiles, value addition broadens consumer options and increases the overall marketability and demand for anchovy-based products. Various drying techniques, such as sun drying and open solar drying, have been employed globally to preserve fish.[25] Solar drying, considered superior to traditional sun-drying methods, produces higher-quality dried fish regarding nutritional value and hygiene, reducing the risk of insect infestation.[26] Additionally, the drying process decreases moisture content, providing a stable protein source for individuals with limited access to fresh fish.[27] Various techniques include washing, drying, and boiling fish in saltwater. The drying process involves either sun-drying or artificial drying, with artificial methods utilizing mechanical or electrical equipment such as radiation drying for efficient moisture removal.[28] Despite technological advancements, some Southeast Asian countries, such as Thailand, Indonesia, and Malaysia, still prefer traditional open-air drying methods to preserve anchovies.[23] [29]

In our study, oven drying at an optimal temperature was opted. Oven drying offers benefits such as a controlled environment, reduced drying time, year-round availability, hygienic conditions, consistent quality, and the preservation of nutritional value. The controlled temperature and humidity settings of the oven contribute to a faster, more reliable drying process compared with traditional methods, particularly advantageous when consistent sunlight is unavailable throughout the year. The controlled environment minimizes contamination risks, ensuring a more hygienic drying process and a compatible, high-quality end product. Moreover, oven drying aids in preserving the nutritional content of anchovies by controlling the drying conditions.[30] The dried anchovy fish was finely ground into a powder using a pulverizer. The resulting fish powder ([Fig. 3]) was then carefully packaged in polypropylene bags and stored at -18°C until used.

Fig. 3 Packed dried anchovy fish powder.

Anchovies are commonly dried and integrated into various cuisines. In Malaysia, for instance, anchovies are promptly washed and boiled in a 10% brine solution upon landing, followed by sun-drying.[23] In Indonesia, dried anchovies are prepared by boiling them in a salt solution (3–4%) followed by air drying.[31] The salting process serves as a pre-treatment, improving the product's ability to dry quickly and reducing its water activity, thereby extending shelf life and altering the sensory characteristics of the food.[32]

However, drying may compromise nutritional quality, leading to vitamin A degradation, lipid oxidation, and protein breakdown.[33] Furthermore, the open-air drying method exposes the product to contaminants, posing hygiene concerns for consumers.[25] Distributing dried anchovy powder offers numerous advantages, including extended shelf life, storage and transportation convenience, culinary application versatility, retained nutritional value, cost-effectiveness, reduced fishy odor, improved ingredient homogeneity, and extended usability. These attributes collectively position dried anchovy powder as a valuable and widely applicable ingredient in the global food market.