A thesis submitted in partial fulfillment of requirements for conferment of the Degree of Masters of Science in Molecular Biology of Meru University of Science and Technology

Fruits and fruit-based products comprise abundant bioactive compounds valuable to human health
and may reduce the risk of disease by beneficially targeting body functions. Consequently, these
fruits are used in varying proportions as ingredients in functional foods. The market sectors for
fruit juices have been growing at a fast pace. The widening market of these products has led to
speculation that they may contain artificial aromas, adulterated and mislabeled. Fruits are
relatively easy to authenticate morphologically when intact and fresh. However, the act of
processing them into juice gives rise to the possibility of substitution with cheaper products. For
this reason, processed food product authentication is primarily significant for consumers;
industries, and regulatory agencies. Effective, reliable, and rapid food authentication methods are
valuable tools for the identification of natural fruit pulp in reconstituted fruit juices to ensure juice
quality and safety hence mitigating adulteration and fraud. Molecular-based methods have recently
acquired immense priority for their ability to pick food material sources at any stage along the food
supply chain. The study focused on DNA isolation from raw and reconstituted fruit juices. The
study aimed to validate an appropriate DNA isolation protocol specifically for processed fruit
juices. It describes an innovative experimental methodology that efficiently extracts, amplifies,
and identifies natural fruit juice pulp by utilizing universal biomarkers to test for the quality and
authenticity of natural fruit pulps in reconstituted fruit juices in Kenyan markets. Two genomic
DNA extraction protocols; CTAB and SDS were tested for the isolation of DNA from processed
fruit juices. The CTAB and SDS methods were able to recover genomic DNA of high quality and
purity appropriate for application in various PCR analyses with few limitations in the CTAB
protocol. The concentration of the DNA was determined using the Nano-drop spectrophotometer
in ng/μL by calculating the absorbance at wavelengths (A260/A280: A260/A230). The quality of
the extracted DNA was evaluated on 0.8% agarose gel electrophoresis stained with lul ethidium
bromide and observation of bands integrity was done in a UV-trans-illuminator machine (Quantum
ST4, France). PCR amplification was done using universal primers (rbcL-650 bp, psbA-323 bp)
that target the plant chloroplast genome). DNA extracted from the SDS method exhibited
robustness and ease during the PCR amplification process. The amplified bands' quality and
integrity were evaluated on 1.5% agarose gel stained with 1 ng/L ethidium bromide. From the
results obtained, the SDS protocol emerged as the best for extracting high-quantity and amplifiable
DNA.
KEYWORDS;
Adulteration, Molecular markers, Fruit Juice, Food Safety, Quality, Protocol, DNA, SDS, CTAB