Optimization and microbial assessment of white leg shrimp

(Litopenaeus vannamei) shell powder

An Undergraduate Thesis

Presented to the Faculty

Member of the

NORTH EASTERN MINDANAO STATE UNIVERSITY

LIANGA CAMPUS

Lianga, Surigao Del Sur

In partial Fullfillment

Of the Requirements for the Degree of

BACHELOR OF SCIENCE IN FISHERIES

Bastida, Juane

Agudo, Bless

MARCH 2024
 CHAPTER I

INTRODUCTION

Seafood processing by-products, like shrimp shells, are rich in valuable

compounds like chitin, proteins, and minerals, making them a promising

resource for various industries (Ipsheta et al., 2023). These by-products can

be utilized in sustainable ways, such as producing biodegradable marine

biopolymers for food packaging (Atif et al., 2023). Additionally, marine raw

materials from seafood waste can be repurposed for developing eco-friendly

plant protection products in agriculture (Aline et al., 2022). The circular

economy concept emphasizes converting waste into wealth, aligning with the

idea of valorizing seafood waste to create value-added products like

biopolymers for food packaging (N. Topic et al., 2023). Therefore, exploring

shrimp shells and other seafood processing by-products can lead to

innovative applications in food, pharmaceuticals, and agriculture, contributing

to a more sustainable and environmentally friendly approach in various

industries.

Shrimp shells are primarily composed of chitin, a biopolymer known for

its versatile properties such as biocompatibility, antimicrobial activity, and

biodegradability (Vaughn & Souza, 2023). Chitin is a polysaccharide found in

the exoskeletons of crustaceans like shrimp, crabs, lobsters, and insects,

providing structural support and protection (Toliba et al., 2014). This

compound is widely used in various applications due to its biodegradability,

making it an environmentally friendly material with potential in fields like

biomedicine, agriculture, and wastewater treatment. Chitin's antimicrobial

properties make it valuable in wound healing and as a natural preservative in

food products (Gharbi et al., 2023). The composition of shrimp shells, rich in

chitin, offers a sustainable and valuable resource with diverse applications

across different industries.

The direct utilization of shrimp shell powder offers a cost-effective and

environmentally friendly approach to harnessing the inherent benefits of this

abundant by-product. Shrimp shells are primarily composed of chitin, a

biopolymer renowned for its versatile properties, including biocompatibility,

antimicrobial activity, and biodegradability (Mathew et al., 2020). These

properties make shrimp shells a valuable resource for various applications,

such as food, pharmaceuticals, and agriculture. Shrimp shells are also a

potential raw material for agrochemicals, microbial culture media, adsorbents,

therapeutics, nutraceuticals, and bio-nanomaterials (Wani et al., 2023). In

addition, the bioconversion of shrimp waste is cost-effective, ecofriendly, and

safe, making it an attractive alternative to traditional chemical processing

methods. Microbial fermentation and the action of exogenous enzymes are

among the significant SW bioconversion processes, which can be used to

produce valuable products from shrimp waste.

This thesis focuses on the optimization and microbial assessment of

white leg shrimp shell powder, aiming to elucidate its potential as a value-

added material in various applications. The powdered shrimp shells offer a

various application not only in the food industry but also can be in agricultural

and pharmaceuticals. Its importance is to decrease and utilized the by-product

of the shrimp shell into value-added products. This thesis aims to highlight

how important it is to turn waste into valuable resources by explaining the

possibilities of powdered shrimp shells in these diverse sectors. It attempts to

offer insights into using the natural qualities of shrimp shell powder for

purposes that are both economically and environmentally feasible through

optimization strategies and microbiological evaluations.

Objective of the Study

To optimize the shell of white legged shrimp and assess its microbes,

the goal of the study are follows:

1. Calculate the yield of the extracted powder out of the white legged

shrimp shell;

2. Conducts a sensory evaluation on the powder shrimps;

3. Conduct sensory profiling on the color, taste, odor, texture, and overall

acceptability on the powdered white legged shrimp shell.

4. Evaluate quality and safety of the powdered shrimp shell through

microbial assessment.

5. Evaluate the economic viability of each treatment.

Significance of the Study

The findings of this research will benefit society by giving consumers

access to things that are equally as unique, high-quality, and nourishing as

those that are sold for a premium but lack sustenance. The study will provide

consumers with additional nutritional value and health benefits, particularly

those who follow a healthy diet and lifestyle. In addition, this study will help

our local fisherfolk to reduce the waste of white legged shrimp and will be

utilized its by product.

Conceptual Framework

The figure 1 will be the flow chart of in conducting this study. White

legged shrimp will act as the top of the paradigm as it signifies the

independent variable in this study. It illustrates the paradigm of the conceptual

framework.

Shrimp Shell

Different Treatments: Shrimp (Litopenaeus

vannamei)
T1 = 25 grams
Source of material:
T2= 50 grams
T3= 75 grams

Yield Sensory Crude Microbial Return of

evaluation Protein Assessment Investment

Appearance Aroma Flavor Texture Acceptability

Figure 1. Paradigm of the Variables on the Optimization and microbial

assessment of Shrimp Shell

Scope and Limitations of the Study

The study will focus on the extraction of white leg shrimp (Litopenaeus

vannamei) shells and the optimization of the extraction process to obtain

shrimp shell powder. The respondents enlisted for sensory evaluation will

encompass trained panelists, students, and individuals selected randomly. To

assess the yield of powdered shell, the product will be weighted.

Definition of Terms

Acceptability - Refers to the likeliness of the

product according to the

consumers sensory evaluation

such as aroma, flavor, texture

and appearance.

Appearance - The physical look of the

powdered shrimp.

Aroma - Refers to the smell of the product

Crude Protein - The amount of protein present in

powdered shrimp shell

Flavor - Refers to the taste

Microbial Assessment - Refers to the testing of the

possible microbes of the

powdered shell.

Optimization - Means of utilizing by-product of

the shrimp such as its shell to

powdered for other consumption.

Powdered - Fine grain texture after being

processed the shrimp shell.

Return of Investment - The profit of the powdered shell

calculated by subtracting the

initial cost of the investment from

its final value, then dividing this

new number by the cost of the

 investment, and finally,

multiplying it by 100.

Sensory Evaluation - Testing the powdered shrimp

such as aroma, appearance,

texture and flavor and interpret

the result.

Shrimp Shell (Litopenaeus - The ingredients used in the

vannamei) powdered shrimp.

Texture - The feel and appearance of the

powdered shrimp shell.

 CHAPTER II

REVIEW OF RELATED LITERATURE

Taxonomy of White Legged Shrimp (Litopenneaus vanamei)

The taxonomy of the whiteleg shrimp, scientifically known as

Litopenaeus vannamei, is as follows (Boone, 1931):

Kingdom: Animalia

Phylum: Arthropoda

Class: Malacostraca

Order: Decapoda

Family: Penaeidae

Genus: Litopenaeus

Species: Litopenaeus

vannamei

Figure 2. whiteleg shrimp (Litopenaeus vannamei)

Biology of Whiteleg Shrimp

Litopenaeus vannamei, also known as the Pacific white shrimp, is a

significant species in global shrimp aquaculture, cultivated extensively for food

consumption (Qianfu et al., 2022). This species exhibits stable production,

high disease resistance, and ease of stocking at high densities, making it a

preferred choice for farmers (Prajapati & Ujjania, 2022).

This species holds significant economic value due to its high market

price, low susceptibility to diseases, and popularity for consumption, making it

a key player in global shrimp aquaculture. Cultivation efforts for Litopenaeus

vannamei focus on enhancing aquaculture performance, with strategies like

the use of probiotics to aid in digestion and disease resistance. Probiotics,

derived from non-pathogenic bacteria, are cost-effective and contribute to

improved survival rates, water quality, immunity, and disease resistance in

shrimp farming operations (Amiin et al., 2023).

Litopenaeus vannamei, commonly known as whiteleg shrimp, thrives in

coastal areas, estuaries, and lagoons with sandy or muddy substrates,

typically in shallow waters, displaying adaptability to various salinities (Briggs

& Fox, 2022). Additionally, L. vannamei a euryhaline shrimp, can adapt to

various salinities, making it suitable for aquaculture in diverse environments,

as indicated in the study on growth performance at different salinities (Fingki

et al., 2023).

The white shrimp can tolerate a wide range of salinities, from 0.5 to 50

practical salinity units (PSU), and can adapt to low salinity environments,

making it suitable for inland aquaculture. The growth performance of the

Pacific white shrimp is influenced by salinity, with optimal growth occurring at

salinities between 15 and 30 ppt. The shrimp's osmoregulatory capacity is

affected by temperature and salinity, with an optimal culture environment for

growth and osmoregulation occurring at temperatures between 28 and 32°C

and salinities between 20 and 40 ppt (Chong-Roblesa et al., 2014). The

shrimp's osmoregulatory capacity is influenced by its ability to regulate

hemolymph osmotic pressure and the development of compensatory

mechanisms for homeostasis. The shrimp's osmoregulatory capacity is

affected by salinity, with a decrease in osmoregulatory capacity observed at

low salinities (Ye et al., 2023).

The life cycle of whiteleg shrimp, consists of several stages including

eggs, nauplius, zoea, mysis, post-larvae, juvenile, and adult stages. The eggs

are laid by female shrimp after spawning with male shrimp, and hatch into

microscopic larvae called nauplii within 12-16 hours. The nauplii undergo six

changes of form, or instars, before metamorphosing into zoea, which are

larger and have a more distinct shrimp-like appearance. The zoea undergo

three changes of form before metamorphosing into mysis, which are even

larger and have a fan-shaped tail. The mysis undergo three changes of form

before metamorphosing into post-larvae, which are the final larval stage and

resemble small adult shrimp. The post-larvae are raised in ponds until they

reach the juvenile stage, which is the first benthic stage and resembles adult

shrimp. The juvenile stage is a vulnerable period in the shrimp life cycle, and

shrimp require a suitable habitat with the right amount of food and water

quality. After 80-90 days from the juvenile stage, the shrimp grow into young

adults and are ready for harvest (Rojo Arreola et al., 2020).
 A prominent shrimp species, undergoes a complex life cycle

encompassing various stages from larvae to adults. The larvae experience

distinct developmental phases before transforming into post-larvae, settling,

and progressing into juveniles and ultimately adults. Studies highlight the

significance of innate immunity during the Zoea larvae stage, suggesting a

defense mechanism against pathogens like Vibrio vulnificus (Chen et al.,

2023). Furthermore, the provision of phytoplankton during larval stages

significantly impacts the survival of vannamei shrimp larvae, with treatment D

showing the highest survival rate (Ratwanati & Ilham, 2022). The assembly of

the larval shrimp bacterial community plays a crucial role in determining the

success of shrimp culture, emphasizing the importance of microbial

management in rearing water for beneficial larval microbiota (Briggs & Fox,

2022).

Litopenaeus vannamei, also known as the Pacific white shrimp, is a

species that is known for its relatively good disease resistance. However, it is

still susceptible to various viral, bacterial, and fungal pathogens that can

cause significant losses in aquaculture operations. Common diseases that

affect this species include white spot syndrome virus (WSSV), infectious

myonecrosis virus (IMNV), and Taura syndrome virus (TSV), among others

(Sivakamavalli et al., 2021). Disease management strategies for Litopenaeus

vannamei include biosecurity measures, vaccination (where available), and

the use of probiotics and immunostimulants to enhance the shrimp's immune

response. Biosecurity measures are essential for preventing the introduction

and spread of diseases in aquaculture operations. These measures include

the use of disinfectants, quarantine procedures, and the management of

water quality and feed (Cock, Salazar, & Rye, 2015).

Distribution and Economic Importance

Whiteleg shrimp, scientifically known as Litopenaeus vannamei, is an

important cultured species that accounts for more than 53% of the total

production of farmed crustaceans worldwide. This species is native to tropical

marine habitats along the eastern Pacific Coast, from Peru to Mexico, and has

been introduced into mainland China in 1988, where it has become the most

widely cultivated shrimp species due to its favorable and efficient culture

characteristics (Kim et al., 2020).

The distribution of whiteleg shrimp holds significant importance in the

seafood industry. In Vietnam, the distribution channel involves middlemen who

play a crucial role in the marketing process, exerting power and influencing

the distribution of shrimp based on size categories to various market

segments (Tram et al., 2016). In Bangladesh, the introduction of Whiteleg

shrimp has shown promising results in boosting shrimp production and

increasing export earnings substantially, indicating its potential to revolutionize

the aquaculture sector Naser et al., 2022). Understanding the distribution

patterns of shrimp species like Litopenaeus vannamei is essential for

aquaculture development, considering factors such as resistance to diseases,

growth indices, and stress tolerance, which are vital for sustainable farming

practices (Tram et al., 2017). Additionally, the distribution of VP28 protein in

Whiteleg shrimp organs post-injection has implications for immunological

responses and disease resistance, showcasing the importance of distribution

in enhancing shrimp health and survival rates (Cabrera-Jimenez, 2013).

Related Studies

Powder Shell

Powdered shrimp shell refers to the finely ground form of shrimp shells,

which can be utilized in various applications. Studies have shown that shrimp

shell powder can be used as a production medium for optimizing fermentation

conditions, leading to the production of bioactive metabolites with

antimicrobial properties against pathogens like Escherichia coli and MRSA

(Ayatollah et al., 2021). Additionally, shrimp shell powder can be processed

into calcined oyster shell powder, which acts as a natural preservative to

improve the shelf-life of aquatic products like shrimp by inhibiting bacterial

growth and reducing spoilage during refrigerated storage (Wen-Chien Lu et

al., 2022). Furthermore, innovative methods have been developed to prepare

shrimp shell powder, ensuring quality preservation and environmental

sustainability through processes like steaming, disinfecting, sterilizing, and

drying (Chen et al., 2016).

Powdered shrimp shell has shown promising applications in various

fields. Studies have highlighted its potential in producing bioactive metabolites

with antimicrobial properties, enhancing the shelf-life of aquatic products as a

natural preservative, and aiding in the efficient degradation of chitin through

the discovery of novel lytic polysaccharide monooxygenases (LPMO) (Yan et

al., 2023). Additionally, research has demonstrated the beneficial effects of

shrimp shell powder in improving Total Antioxidant Capacity (TAC) and

reducing Homeostatic Model Assessment-Insulin Resistance (HOMA-IR)

levels in diabetic conditions (Risya Ahriyasna et al., 2021). Furthermore, the

utilization of Litopenaeus vannamei shell powder has been linked to increased

adiponectin levels, improved lipid markers, and reduced cardiovascular

disease risk in type 2 diabetes mellitus (T2DM) rats (Annisa et al., 2019).

Powdering shrimp shells aids in chitosan extraction by demineralization

and deproteinization processes, facilitating the synthesis of chitosan

nanopowders for various applications due to its unique properties (Tonmoy et

al., 2022). Powdered shrimp shells are utilized to enrich shrimp crackers with

protein, reducing waste and creating a high-quality, cost-effective product

beneficial for protein fortification and potential malnutrition alleviation

(Murshida & Nowsad, 2013).

Utilization of Shrimp Shell

Shrimp shells have diverse applications, showcasing their potential

beyond waste. Studies highlight their use in producing biopolymers with

enhanced properties like antimicrobial activity (Topic et al., 2023). Additionally,

shrimp shells can serve as a medium for cultivating marine Streptomyces,

yielding bioactive metabolites and chitinase enzymes with antimicrobial

effects (Aishwarya et al., 2022). Furthermore, shrimp shell waste can be

transformed into shrimp flour, rich in protein, fat, carbohydrates, and ash,

making it a viable alternative for marine fish feed production. Moreover,

shrimp shells can be utilized as an effective adsorbent for heavy metals like
cobalt in water treatment processes, displaying comparable efficiency to other

adsorbents (Gök et al., 2022).

Shrimp shells can also be processed into shrimp flour, which can be

used as a source of minerals in the preparation of fish feed rations. The ash

content in shrimp shell flour is the highest among all other nutrients, making it

an excellent source of minerals. Shrimp shell flour can also be used as a

source of protein, fat, and carbohydrates in the preparation of fish feed

(Yulianto et al., 2021).

Another study found that shrimp shell waste can be processed into

chitosan, a new material with economic value and environmental benefits.

Chitosan is a biodegradable and biocompatible material with various

applications, including water treatment, food packaging, and pharmaceuticals.

The study found that shrimp shell waste can be processed into chitosan

through deproteinization, demineralization, and deacetylation processes

(Zulkarnain et al., 2023).

Powder Shrimp Shell as Food

Powdered shrimp shell can be utilized in various food applications due

to its nutritional and functional properties. Studies have explored the use of

shrimp shell powder in different food products, such as seafood seasoning,

extruded snacks, and as a natural preservative for aquatic products

(Manikandan et al., 2020). The powder extracted from shrimp shells has been

found to be rich in protein, antioxidants, and bioactive peptides, making it a

valuable ingredient for fortification and enrichment in food processing (Keshk

& Emara, 2018). Additionally, the development of edible fish powder from
mantis shrimp has shown promising results in enhancing the nutrient content

of food products like wheat sticks and potato croquettes.

Powdered shrimp shell can be used as a food ingredient, particularly

as a flavoring agent. It can be prepared by refluxing shrimp shells in distilled

water with added salt and enzymes, such as bromelain, to extract the flavorful

compounds. The resulting filtrate can be further processed into a powder

form, which can be used in various dishes, such as soups, stews, and sauces,

to add a rich shrimp flavor (Rengga et al., 2019).

Superfine shrimp shell powder is used in high-calcium sausage,

providing nutritional value, unique flavor, texture, and calcium

supplementation, meeting national standards for a health-conscious meat

product (Chen et al., 2014).

 CHAPTER III

MATERIALS AND METHOD

Materials and Ingredients

In this study, researchers will used grinder used for grinding the shrimp

shell, oven for drying the grind shrimp shell, mesh screen to sieve the grind

shrimp shell to have uniform particles size and separate the larger particles

from the desired finer powder, digital weighing scale to weighs the shrimp

shell before and after grinding, and container to store the shrimp shell and

prevent from the contamination of bacteria and other microorganisms. The

ingredients use will be the Shrimp Shell mainly the Litopenaeus vannamei,

ensuring the shrimp shells will be used is clean and free from contaminants

before processing.

Experimental Site

The research will be carried out at the Lianga Campus of North Eastern

Mindanao State University (NEMSU), Lianga Surigao del Sur. Testing for

sensory evaluation and microbial assessment will be done in the office of the

Bachelor of Science in Fisheries. Random individuals outside the institution

will perform the test in the same location. The researcher made sure that

there were suitable and safe laboratory facilities.

 Figure 3. location of the study site, NEMSU-Lianga Campus.

Data Gatherings and Procedures

Preparation of Shrimp Shell

Collect fresh shrimp shells as by-products from seafood processing or

cooking. Rinse the shells thoroughly under running water to remove any dirt,

debris, or residual meat. then, place the shrimp shell for weighing scale for the

desired amount to be used in each treatment.

Experimental Design

This study will employ the Completely Randomized Design (CRD)

methodology. Analysis of variance (ANOVA) and Duncan's multiple range test

will be used to assess the data. The sensory evaluation exam will be

conducted using the hedonic test approach. The experiment was consisting of
3 treatments with 3 replications 25 grams, 50 grams, and 75 grams of shrimp

shell. The treatment and replication shown in table below.

Table 1. treatments and replication of the study. Treatment (TR) and Replicate

(R).

Treatments Shrimp Shell (grams)

TR1 (R1) 25
TR1 (R2) 25
TR1 (R3) 25

TR2 (R1) 50
TR2 (R2) 50
TR2 (R3) 50

TR3 (R1) 75
TR3 (R2) 75
TR3 (R3) 75

The table below represents the flow of the production of powdered

shrimp shell and data gathering.

 Testing
Shrimp Shell

Sensory Microbial
Collecting
Evaluation Assessment

Cleaning
Appearance

Weighing
Aroma

Drying
Texture

Grinding Flavor

Sieving Powder

Figure 4. the flow of this study.

Yield (Swastawati et. al., 2022)

The yield of the powdered shrimp shell will be measured through

calculating the yield before and after it is powdered. The yield was based on

the ratio of the weight shrimp shell before grinding and the weight of

powdered shrimp shell as determined in the formula below:

Shrimp shell weight

Yield (%)= x 100%
powdered shrimp shell weight

Sensory Testing

Participants will be instructed to engage in a sensory evaluation by

reading and completing an evaluation form. This will involve tasting samples
from each of the three measurement levels, which will be presented

simultaneously, in any sequence, and with a random combination of three

numbers printed on them. Each participant will receive a set of three separate

treatments, stapled together, with the control number placed on the top sheet.

Participants will be using a hedonic scale in order to rate each sample's

taste, flavor, appearance, scent, and overall acceptability. The sensory forms

arranged on the table will serve as their guide, instructing them to taste the

samples in that order. The respondents will write additional remarks on the

underside of the sensory instrument.

Sample Collection

The powdered shrimp shell will undergo microbial assessment to

assess the quality of the product being utilized as value added product. There

will be 3 samples weighing 10 grams each will be used for microbiological

analysis. In total, there will be 3 samples with a total weight of 30 grams.

Crude Protein Analysis

The crude protein analysis involved three (3) treatments. Each

treatment will be appropriately labeled. The powdered shrimp shell will be put

in a sealable container like glass to prevent contamination with labeled, the

labeled treatments, along with their glass jars. This will examine the amount of

protein in powdered shrimp shell for healthier consumptions.

Data Gathering

The sensory qualities and overall acceptance of the finished product for

each of the three treatments will be evaluated by the researchers in this study.
Questionnaires will also be used to assess the sensory qualities. The hedonic

exam will involve 45 responders in all, including faculty, students, and people

chosen at random. The questionnaire results will be totaled, tabulated, and

scored in preparation for analysis.

Cost and Return Analysis

Cost and return analysis through the assumption of each treatment will

be computed to determine the economic feasibility and profitability of the

culture scheme. Among on important economic equations are as follows:

Net income = gross sales – production cost

Net Income
Return on investment (ROI) = x 100 %
production cost

gross income
Benefit- cost ratio (BCR)=
total cost of production

Data Analysis

An Excel spreadsheet with the data will be used to perform statistical

calculations. Finding the means and standard deviations for texture, flavor,

scent, and appearance will be part of the analysis. Analysis of Variance

(ANOVA) will be used to determine statistical significance, and Tukey's

Pairwise test will be utilized for post-hoc comparisons to identify mean

differences.
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