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BIOENZYMATIC ACTIVITY OF KARISKIS (Albizia Lebbekoides) DURING THE FERMENTATION OF SUKANG ILOCO

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A Research

Presented to
The Teachers of the Science, Technology Engineering and Mathematics (STEM)
Senior High School
Ilocos Sur National High School
Vigan City, Ilocos Sur

______________________________

In Partial Fulfilment of the
Requirements for the Subject
Research/Capstone Project

_____________________________

by:

KRISHA ANNE S. AGCAMARAN
NEHEMIAH G. RABENA
ANGEL JOY C. RAPACON
JONALD A. BAUTISTA

2017
Chapter I
INTRODUCTION

Background of the Study

Vinegar, or Suka (as it is known in the Philippines), is one of the most used ingredients in the Filipino kitchen. The prevalent use of Suka is due to its large part in prolonging the shelf life bestowed upon the cooked foods. But aside from its preservative treatment, Filipinos also just happen to like the elevated flavor punch that it provides.
Particularly, Ilocano people are fun in eating foods with condiments. One of these condiments is vinegar especially Sukang Iloko which was originated in the province of Ilocos Sur. Sukang Iloko is special because of its great taste, aroma and presentation.
The manufacture of Ilocos vinegar (Sukang Iloko) is traced long before Spain colonized the Philippines. Although this product was already part of a flourishing trade between the Philippines and its neighboring countries, Ilocos vinegar in particular reached prominence when it became an export via the Galleon Trade to Europe by way of Acapulco, which lasted for nearly two centuries.
According to the synopsis of the Basi Revolt, the significance of the industry during the Spanish period can be attested by the fact that the Spanish government declared monopoly on the industry in the same way the Tobacco Monopoly was imposed. This move stirred an uprising by the brewers and natives in the Ilocos region which became known as the Basi Revolt of 1807.
However, the Commonwealth era further saw the decline in the production of sukang Iloko displaced by imported products. This was exacerbated by the outbreak of the World War II. Today’s generation has a vague idea of this fine, traditional industry, which was once the pride of the ancestors, particularly the Ilocanos.
At the present time, Ilocos Vinegar is well-known even outside the province of Ilocos Sur. Due to this, its production rate is high. In this condition, the rate of usage of the fermentation agent to be used to ferment the Sukang Iloko is also at high quantity. One of the fermentation agent that are used by the manufacturers is the Albizia Lebbekoides which is called Kariskis by the Filipinos.
Albizia Lebbekoides is a plant that is widely distributed in South-East Asia, where the species is found in Thailand, Laos, Cambodia, Vietnam, the Philippines, southern Sulawesi, Java and the Lesser Sunda Islands.
Albizia Lebbekoides is a tree that is usually growing 8 to 15 meters tall but sometimes up to 32 meters with a trunk of 40 cm up to 80 cm in diameter. Its Bark is smooth, grey or greyish brown. The Branches sometimes drooping, with a dense foliage and its living bark is beefy red. Branchlets are terete, glabrous (or tomentellous). Stipules are filiform, subulate, with a length 1.5-2 mm, early caducous. Leaves are alternate, minutely stipulate, bipinnately compound with 5-13 cm long rachis provided with glands near base and top; petiole 2.5-6 cm long; pinnae in 3-8 pairs, with glandular axis, 5-15 cm long; leaflets (5-)15-25(-35) pairs per pinna, (narrowly) oblong, 6-20 mm × 2-6 mm, asymmetric and truncate at base, mucronate at apex, sessile.
The inflorescences are peduncled or five fascicled, terminal, or axillary. The flowers are bisexual, sessile, uniform, pentamerous and fragrant. Each flower has a white and green calyx. Strap-shaped dehiscent pods are dark brown when ripe, flat straight or slightly curved, oblong, and 8.5-15 centimeters long. The Number of seeds reaches up to 12 per pod.
It occurs commonly in deciduous forests in dry localities, less commonly in savanna and evergreen forests. The species prefers open locations, such as forest margins, road-sides, along streams, and in forest clearings. More rarely it is found in shaded habitats. The species grows from sea-level to an altitude of 800 m, both on red volcanic soil and limestone.
In Java the bark is occasionally and locally used to tan hides and fishing nets. Moreover, it provides a red dye, formerly used for coloring cloth and known as “soga tekik” in eastern Java. In the Philippines it is frequently used in the manufacture of a fermented drink made from sugar cane, just like the bark of Macaranga tanarius which also yields tannin. The timber is suited for indoor construction, and in Cambodia paddy mills are constructed from the wood. The bark is used medicinally just like many other tannin-yielding barks as a remedy for colic in Cambodia. It is also sometimes planted as a shade tree.
Use of tannic acid in food applications is far more widespread and significant amounts are used as process aids in beer clarification, aroma compound in soft drinks and juices. Equally important are applications in the wine industry, where it finds use as a natural clarifying agent, color stabilizer and taste enhancer.
Vinegar is commonly used as food ingredient but also for its medicinal properties and for its physiological effects such as invigorating, regulator of blood pressure, diabetes mellitus regulator, appetite stimulator, digestion and absorption of calcium (Ndoye et al., 2007). Consequently, acetic acid bacteria cause an important industrial interest as well as lactic acid bacteria and yeast. Since, the acetic bacteria are involved in the production or spoilage of food, their species identification is lead information for the technologist trying to control a bioprocess in industry (Trcek, 2005).
In recent years, there have been major advances in understanding their taxonomy, molecular biology and physiology and in methods for their isolation and identification (Raspor and Goranoviccaron, 2008). However, problems related to environment conditions such as temperature variations and process technology limit the artisanal and industrial applications in tropical regions (Ndoye, et al., 2007).The production of traditional balsamic vinegar uses a selection of yeasts and acetic bacteria (De Vero, et al., 2006).
Ndoye et al. (2006) reported that acetic bacteria are gram-negative strictly aerobic bacteria and commonly found in nature on vegetable products (fruits, cereals, herbs). They have the ability to oxidize the different types of alcohol in major food products derived from biotechnology as vinegar. The strains involved in production of high acetic degree vinegar are rarely isolated from the environment with success. Even after the successful isolation, the strains have proven to be less efficient and extremely difficult for handling under laboratory conditions, especially if the idea is to preserve their original high acetic acid resistance (Treck, 2005).
A few species of acetic bacteria are able to grow at high sugar concentration, but the sugar tolerance is an important acetic bacteria trait for traditional Balsamic Vinegar production, since this product is special vinegar made from cooked must with a high sugar concentration (Gullo, et al., 2005).
Vinegar is the product made from the conversion of ethyl alcohol to acetic acid by a genus of bacteria, Acetobacter. Therefore, vinegar can be produced from any alcoholic material from alcohol-water mixtures to various fruit wines (Peppler and Beaman, 1967).
The researchers found out that in the absence of the mother agent, some utilized a Kariskis bark when this revolt occurred. For this case, the researchers would like to find out if Kariskis bark can be utilized as a reactive enzyme in the production of Sukang Iloco.
The idea of reviving this sunset industry holds potentials in creating livelihood opportunities, and in integrating agriculture and industry in the classical concept of agribusiness that is rural- and people-based. The industry offers natural products that protect people’s health, and are friendly to the environment. Lastly it revives the spirit of nationalism, culture and tradition.

Statement of the problem
This study was conducted to discover the bio enzymatic activity of Kariskis bark (Albizia Lebbekoides) during the fermentation of Sukang Iloco:
1. What is the physical property of the Kariskis (Albizia Lebbekoides) that is helpful in the fermentation process of Sukang Iloko?
2. What are the methods/procedures to be used to utilize the bioenzymatic activity of Kariskis (Albizia Lebbekoides) on the fermentation of Sukang Iloko?
3. Is there an effect of the Kariskis bark (Albizia Lebbekoides) on the bio-enzymatic fermentation of Sukang Iloco in terms of:
a. taste,
b. smell,
c. appearance, and
d. acidity
4. Is there a significant difference between the Bio Enzymatic Activity of Kariskis (Albizia Lebbekoides) and without the Bio Enzymatic Activity during the Fermentation of Sukang Iloco in terms of :
a. taste,
b. smell,
c. appearance, and
d. acidity
5. Can the Bio Enzymatic Activity of Kariskis (Albizia Lebbekoides) be possible in improving the fermentation process of Sukang Iloco.

Significance of the Study
Since Sukang Iloco is a versatile liquid use in some preparation, as an ingredient and condiment in the province of Ilocos Sur, the concept of bio enzymatic activity brightens the researchers to find an alternative way on improving the fermentation process of the Sukang Iloco.
This study aims to look for alternative method to ferment the Sukang Iloco better and faster. The study will be helpful for the manufacturers of Sukang Iloco for them to ferment the Sukang Iloco in shorter period of time. Thus, it will help them to manufacture more products in certain period of time.
Scope and Limitation
This study utilized the barks of Kariskis (Albizia Lebbekoides ) in making Sukang Iloco.
The researchers conducted the study for six weeks in the Sto Domingo Ilocos Sur where this is one of the residence of a researcher of this investigation process. The researchers choose this place because it is near to San Ildefonso, where the people are making vinegar. The researchers solved the problem by using their senses and checking the product every week to see the differences or similarities. This would really help them to get the results of the problem, and the most important, to make the investigatory project a successful one.

Assumptions
This study was premised on the following assumptions:
1. The instruments used for the data gathering are valid and measurable.
2. The methods and procedures used are the most appropriate to utilize the bioenzymatic activity of Kariskis (Albizia Lebbekoides) on the fermentation of Sukang Iloco.
3. The variables included as one of the essential content of the study are enough bases to determine the essential of Kariskis bark in the fermentation of Sukang Iloco.
Hyphothesis
This study was set to the following hypothesis:
1. The bark of Kariskis (Albizia Lebbekoides) has the potential to lessen/shorten the time of fermentation of Sukang Iloco.
2. The bio enzymatic activity of Kariskis is essential in the fermentation of sukang Iloco.
3. Kariskis (Albizia Lebbekoides) gives better appearance, taste and aroma during the fermentation of Sukang Iloco.
4. There is a significant difference between the Bio Enzymatic Activity of Kariskis (Albizia Lebbekoides) and without the Bio Enzymatic Activity during the Fermentation of Sukang Iloco in terms of appearance, taste, aroma and acidity.
Operational Definition of Terms
The following terms are operationally defined:
Fermentation- It is the process where in substance converts into another substance in the form of acetic acid.
Bio enzymatic Activity- It is the process utilized to prove the presence in the reaction of fermentation.
Sukang Iloco- It is the product used in the activity. Cane vinegar are made from sugar cane juice and one of the most popular in the Philippines, particular in the Ilocos Region of the northern Philippines.
Kariskis – It is a tree that is usually growing 8 to 15 meters tall but sometimes up to 32 meters with a trunk of 40 cm up to 80 cm in diameter. It belongs to the Albizia species. Its scientific name is Albizia Lebbekoides.

Chapter II
Related Literature and Studies
This section presents the review related literature and studies involving to the current research study. This portion of research contains the following areas related to the study specifically, bio enzymatic activity, vinegar, fermentation, Kariskis and the benefits of Vinegar.
On Bio enzymatic
Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical reactions. The molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life (Berg et. al 2002).
In adition, metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and a new field of pseudo enzyme analysis has recently grown up, recognizing that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual ‘pseudo catalytic’ properties.
According to Schomburg (2013), enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins, although a few are catalytic RNA molecules. The latter are called ribozymes. Enzymes’ specificity comes from their unique three-dimensional structures.
Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5′-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. (Wolfenden, 1995; Callahan & Miller, 2007)
Furthermore, Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme’s activity decreases markedly outside its optimal temperature and pH.
According to Anfinsen (1973), enzymes are generally globular proteins, acting alone or in larger complexes. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Although structure determines function, a novel enzymatic activity cannot yet be predicted from structure alone. Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity. Enzyme denaturation is normally linked to temperatures above a species’ normal level; as a result, enzymes from bacteria living in volcanic environments such as hot springs are prized by industrial users for their ability to function at high temperatures, allowing enzyme-catalysed reactions to be operated at a very high rate.
On biological function of enzymes, according to Mackie (1990), an important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulose, to break down the cellulose cell walls of plant fiber.
In Addition, Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolyzing ATP to generate muscle contraction, and also transport cargo around the cell as part of the cytoskeleton. Other ATPase’s in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.
While on Industrial applications, enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed “from scratch” to catalyze reactions that do not occur in nature. (Berglund, et. al. 2003)

On Vinegar
Vinegar is the world’s oldest cooking ingredient and food preservation method. According to the Vinegar Institute (2005), vinegar’s use can be traced back over 10,000 years. In fact, flavored vinegars have been manufactured and sold for almost 5,000 years. The wide variety of vinegars available today is nothing new. Until the six century BC, the Babylonians were making and selling vinegars flavored with fruit, honey, malt, etc. to gourmets of the time. In addition, the Old Testament and Hippocrates recorded the use of vinegar for medicinal purposes (Kehrer 1921; Conner 1976).
It is a product made from the conversion of ethyl alcohol to acetic acid by a genus of bacteria, Acetoba cter. Therefore, vinegar can be produced from any alcoholic material from alcohol-water mixtures to various fruit wines (Peppler and Beaman 1967). Its color and aroma are greatly dependent on the material from which it is made (Kehrer 1921).
According to Tan (2003), vinegar may be defined as a condiment made from various sugary and starchy materials by alcoholic and subsequent acetic fermentation. Vinegar can be produced by different methods and from various raw materials. Wine (white, red, and sherry wine), cider, fruit musts, malted barley, or pure alcohol are used as substrates. Vinegar production ranges from traditional methods employing wood casks and surface culture to submerged fermentation in acetators (Morales et al 2001).
Vinegar traditionally has been used as a food preservative. Whether naturally produced during fermentation or intentionally added, vinegar retards microbial growth and contributes sensory properties to a number of foods. The wide diversity of products containing vinegar (sauces, ketchup, mayonnaise, etc.) and the current fall in wine consumption have favored an increase in vinegar production (De Ory et al 2002).
Acetic acid is the predominant flavoring and antimicrobial component in vinegar. The following review will focus on the importance of acetic acid as a direct food additive or more recently as a food processing aid, to decontaminate food prior to distribution and consumption (Marshall et al 2000).
Earlier processes used for making vinegar were the Orleans process (which is also known as the slow process), the quick process (which is also called the generator process), and the submerged culture process. The quick process and submerged culture process were developed and are used for commercial vinegar production today.
Vinegar bacteria, also called acetic acid bacteria, are members of the genus Acetobacter and characterized by their ability to convert ethyl alcohol, C2H5OH, into acetic acid, CH3CO2H, by oxidation as shown below;
Anaerobic Aerobic
2C2H5OH ? 2CH3CHO ? 2CH3CO2H + 2H2O
Most bacteria strains derived from vinegar factories are able to oxidize acetic acid to CO2 and H2O (over-oxidation) and therefore are classified in the genus Acetobacter (De Ley et al 1984).
Common types of vinegar include white distilled vinegar, cider vinegar, wine vinegar, rice vinegar, and malt vinegar. Further processing of vinegar, following substrate conversion to acetic acid may include filtration, clarification distillation and pasteurization at 165.2°F (74°C) before it is bottled. Regulations in the United States require vinegar to contain at least 4% acetic acid resulting from acetic acid 3fermentation of ethanol containing substrates.
Labels identifying the diluents used to meet the listed concentration of acid are also required. Acetic acid concentration in vinegar may be expressed using the term “grain”. For example, 100 grain distilled vinegar is a 10% acetic acid solution (Nickol 1979). If higher concentration of acetic acid is required, the dilute solution of acetic acid maybe heat distilled or frozen to slush. The slush is centrifuged to isolate the liquid portion (Nickol 1979; Ebner 1982). Concentration from 10-30% may be achieved using this technique (Chukwu and Cheryan 1996).
Vinegar plays an important role in salad dressings, ketchup, hot sauce and other sauces. This need demands industrial fermentation systems capable of producing a large amount of vinegar. These systems must maintain reliable controls and optimum conditions for acetic acid bacteria fermentation (De Ory et al 1999). Many techniques have been developed to improve industrial production of vinegar. Most try to increase the speed of the transformation of ethanol into acetic acid in the presence of the acetic acid bacteria (Tesfaye et al 2002). Today, the most common technology for the vinegar industry is based on the submerged culture (Hormatka and Ebner 1951) with diverse technical modifications which try to improve the general fermentation conditions (aeration, stirring, heating, etc.).
Vinegar is usually described in terms of grain strength, the grain being ten times the acid percentage. For example, 10% acid is referred to as 100 grain (Cruess 1958). According to the Crisco Company (2005), vinegar varieties vary greatly from country to country.
Vinegar production methods can range from traditional methods employing wood casks (Orleans Process) and surface culture (Generator Process) to submerged fermentation (Morales, et al 2001). Vinegar is an important ingredient in many food products. The need for large amounts of the vinegar demands industrial fermentation systems that are capable of producing volumes that are reliably controlled (De Ory, et. al 1999). Many technical devices have been developed to improve the industrial production of vinegar. Generally, these improvements increase the speed of the transformation of ethanol into acetic acid in the presence of acetic acid bacteria (Tesfaye, et. al 2002).
On Kariskis
According to Mazza (2004), the species is native to Cambodia, East Timor, Indonesia (Java, Lesser Sunda Islands, Sulawesi), Laos, Malaysia, Myanmar, Papua New Guinea, Philippines, Thailand and Vietnam where it mainly lives in the deciduous forests in hilly zones, at the margins of clearings, paths and water streams, in climates characterized by one long dry season.
Moreover, the genus is honored to the Italian nobleman Filippo degli Albizzi who, in 1749, introduced in Florence from Constantinople the Albizia julibrissin; the specific name is the combination of the name of the species Albizia lebbeck (L.) Benth. (1844) and of the Greek suffix ” -??????” (-oeidés), from “?????” (êidos) = shape, look, with reference to the resemblance of the two species.
Its common names are the following: châmri:ëk, kântri:ek (Khmer); haluganit, maganhop-sa-bukid (Tagalog); kedinding, tarisi, tekik (Indonesian); h’uung, kh’aang (Laos); koko, siris (Malaysian); anya kokko (Burmese); kungkur (Singaporean); chamari dong, chamari pa, kang (Thai); b?n xe tr?ng, câm-trang, mu?ng trúc, song râ, xúa (Vietnamese).
The Albizia lebbekoides is an unarmed deciduous tree up to 40 m tall, but that in cultivation keeps lower, with erect trunk up to 40-60 cm of diameter with wrinkled greyish to reddish brown bark; like other Leguminosae the rooting apparatus is able to fix the atmospheric nitrogen enriching the soil.
The leaves, on a 3-6 cm long petiole, are alternate, bipinnate, 5-15 cm long, with 3-8 pairs of paripinnate leaflets, 5-12 cm long, each one composed by 5-30 pairs of opposite sessile leaflets, oblong with asymmetric base, 0,8-2,5 cm long and 0,2-05 cm broad. The terminal or axillar inflorescences are up to 18 cm long panicles, formed by dense capitula of about 1 cm of diameter bearing 10-15 hermaphroditic sessile flowers, 0,8-1 cm long, with greenish penta-parted calyx, about 1,5 mm long, and tubular yellow-greenish penta-parted corolla and several white stamina, 0,6-1 cm long, merged at the base to form a tube.
The fruit is an oblong dehiscent legume, flat, 7-15 cm long and 1,5-2,5 cm broad, of yellowish brown colour containing up to 12 almost circular, flat seeds, 0,7 cm long, 0,5 cm broad and 0,1-0,2 cm thick, of brown colour with a thin U-shaped line (pleurogram) on the sides.
It reproduces by seed, previously scarified and kept in lukewarm water for one day, in draining loam maintained humid at the temperature of 23-26 °C, with germination times of 3-5 days.
Cultivable in the tropical and subtropical regions in full sun on even poor soils, is utilized as shade tree in the tea plantations, for the consolidation of the soils, in the reforestation and as ornamental in the road trees. The wood, of brown color and of good characteristics, is utilized in the constructions and for realizing furniture and fixtures and as fuel; from the bark, rich of tannins, they get a dye and in the Philippines is used for aromatizing a drink obtained from the fermentation of sugar cane. In the study of Tasnin (2014), stated that the phytochemical screening of the bark of Albizia lebbekoides showed positive test for tannin, alkaloid, flavonoid, terpenoid, phlabotannin, saponin, steroid and cardiac glycoside. Locally, the bark is utilized in the traditional medicine as a remedy in colic.
On fermentation
Fermentation is an age-old process naturally processed by microbes and humans have been controlling the process for their benefits. The process of fermentation enriches the flavor of food and preserves it naturally because of secondary metabolites secreted by microbes. It is now well established that lactic acid bacteria (LAB) plays a vital role in fermentation process and they are considered as safe organisms hence assigned GRAS (generally regarded as safe) by WHO (2011).
It is an economical process which increases the nutritional value of the food thereby increasing the health benefits (Hutkins, 2006). Wide varieties of fermented foods are available across the world and are categorised as bean based, grain based, vegetable based, fruit based, dairy based, fish/meat based, tea based and so on.
Moreover, fermentation is process where microbes oxidize complex organic compounds like carbohydrate into simple substances where organic acids act as electron acceptor. The microbes digest the food substrate with its enzymes, increase the flavour, aroma, and texture, make the food edible, enrich the food with vitamins, essential amino acids and above all preserve food naturally.
Fermented foods are produced across the world using various techniques, raw materials and more. However, they fall in four categories namely alcoholic, lactic acid, acetic acid and alkaline fermentation (Soni and Sandhu, 1990). Alcoholic fermentation involves the production of ethanol as the end while lactic acid and acetic acid fermentation produce respective acids as end products. The alkaline fermentation is not well known but widely consumed in South east and African countries. In alkaline fermented foods, the proteins are broken into amino acids and peptides releasing ammonia during fermentation resulting in alkaline pH of the food which is achieved spontaneously by mixing bacteria, especially Bacillus subtilus.
A typical example for alkaline fermented food is Japanese natto (Wang and Fung, 1996). Among these, lactic acid fermentation is widely usedfor preservation of foods. This process is achieved by lactic acid bacteria which includes Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Weissella, Aerococcus, Carnobacterium, Oenococcus, Sporolactobacillus, Teragenococcus, and Vagococcus. Because of their presence in food and contribution of healthy microflora for animals, they are ‘generally recognized as safe’ (GRAS) (Donohue, 2004).
On Benefits of Vinegar
A variety of natural vinegar products are found in civilizations around the world. A review of research on these fermented products indicates numerous reports of health benefits derived by consumption of vinegar components. Therapeutic effects of vinegar arising from consuming the inherent bioactive components including acetic acid, gallic acid, catechin, ephicatechin, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid cause antioxidative, antidiabetic, antimicrobial, antitumor, antiobesity, antihypertensive, and cholesterol-lowering responses.
According to Institute of Food Technologists (2014), the earliest known use of vinegar dates back more than 10,000 years ago and has been used as a food and medicine. A new review article reports on recent studies showing different types of vinegars that may benefit human health. Functional therapeutic properties include beneficial effects on cardiovascular health and blood pressure, antibacterial activity, reduction in the effects of diabetes and increased vigor after exercise. In addition, a few studies showed that people who consumed certain types of vinegar daily may have a decreased appetite.
Moreover, in the study of Chen, et.al (2016) stated that both grain vinegars and fruit vinegars, which are fermented by traditional methods, possess a variety of physiological functions, such as antibacterial, anti-infection, anti-oxidation, blood glucose control, lipid metabolism regulation, weight loss, and anticancer activities. The antibacterial and anti-infection abilities of vinegars are mainly due to the presence of organic acids, polyphenols, and melanoidins. The polyphenols and melanoidins also provide the antioxidant abilities of vinegars, which are produced from the raw materials and fermentation processes, respectively.
In addition, the blood glucose control, lipid metabolism regulation, and weight loss capabilities from vinegars are mainly due to acetic acid. Besides caffeoylsophorose (inhibits disaccharidase) and ligustrazine (improves blood circulation), other functional ingredients present in vinegars provide certain health benefits as well. Regarding anticancer activities, several grain vinegars strongly inhibit the growth of some cancer cells in vivo or in vitro, but related functional ingredients remain largely unknown, except tryptophol in Japanese black soybean vinegar. Considering the discovering of various functional ingredients and clarifying their mechanisms, some vinegars could be functional foods or even medicines, depending on a number of proofs that demonstrate these constituents can cure chronic diseases such as diabetes or cardiovascular problems.

Conceptual Framework
To illustrate the study, a paradigm is used to determine the bio- enzymatic activity of kariskis (Albzia Lebbekoides) during the fermentation of sukang Iloco, illustrated by the input- process- output model is exhibited in figure 1.
Input Process Output

As shown in Figure 1, the input is sugar cane, mother vinegar and the kariskis (Albizia Lebbekoides) barks. For the process, the sugar cane is extracted to get its juice. The sugar cane juice is heated for one hour and poured into the sterilized bottles. The mother vinegar and the kariskis (Albizia Lebbekoides) barks are then added to the sugar cane juice for the fermentation. It is fermented for 6 weeks and as for the result, vinegar is produced.

CHAPTER III
Methodology
The methodology provides a detailed description that elaborates the plan of action, process, or design that informs the methods employed in the study.
In particular, this chapter discusses the context of the study, the methodology employed, specific methods and data collection techniques, step-by-step procedures followed and data analysis technique.
A. Materials and Equipment
Within the conduct of the study, these materials were used by the researchers: sugar cane, kariskis bark and mother vinegar.
In addition, the following equipment were used by the researchers in conducting the study namely: weighing scale, funnel, and measuring cups. The other essential materials and equipment which will be used by the researchers will be visible and readily available along the locality and on their own households.
B. Research Diagram/Flow Chart/Research Design

C. General Procedures / Treatment
1. Preparation of Materials Needed
The researchers gathered ten 1.5 liters of plastics bottles and were cleaned properly. The, kariskis (Albezzia Lebbekoides) bark were also collected. The sugar cane and mother vinegar were acquired in Pusuak, Sto. Domingo Ilocos Sur. The weighing scale, funnel and measuring cups were also prepared by the researchers
2. Experiment Proper
2.1 Preparation of Gathered Materials
First of all, prepare and gather all essential materials, ingredients and equipment needed. They were arranged on a table to have a more organized working area. The ingredients were dried kariskis bark (Albizia Lebbekoides) and mother vinegar. The ingredients were properly stored to a container to avoid them from being wasted.
2.2 Preparation of the Kariskis Bark
The kariskis bark was broke down into smaller pieces and weighed them to acquire the corresponding weight needed in each treatment. Two treatments needed one- fourth kilograms (1/4 kg) of kariskis bark while the other two treatments needed one- eight kilograms (1/8 kg) of kariskis bark in each of them.

2.3 Preparation of Sugar Cane Juice
The sugar cane was gathered through the help of one of the vinegar producer in Sto. Domingo, Ilocos Sur. After gathering, it was extracted to get its juice. The sugar cane juice was heated in an hour. Then it was place into a large container for it to be cooled down.
2.4 Preparation of Mother Vinegar
The mother vinegar used was acquired in the Sto. Domingo, Ilocos Sur. The mother vinegar was measured using measuring cups and each of the treatments needed one hundred milliliters (100 ml) of it. Mother vinegar was put in three treatments specifically on treatment number two, four and treatment number six.
2.5 Preparation of Sukang Iloko
A total of ten 1.5 litters of plastic bottles were fetched from six (6) different treatments. The sugar cane extract was put in the plastic bottles through the use of a strainer and a funnel with its own corresponding ingredients in per treatment.
3. Preparation of the Treatments
There were six (6) treatments in the experiment and each of them has two (2) samples. One kilogram (1 kg) of the cooked sugar cane juice was place in each treatment.
Kariskis barks were put on the four (4) treatments with the desired amount specifically on treatments number 3 to number 6. One hundred milliliters (100 ml) of mother vinegar was also put on three (3) treatments specifically on treatment number 2, 4 and 6.
For every treatment, the following were used on the study.
Treatment Components
1 One kilogram (1 kg) of sugar cane juice
2 One kilogram (1 kg) of sugar cane juice with one hundred milliliters (100 ml) of mother vinegar
3 One kilogram (1 kg) of sugar cane juice and one hundred twenty five milliliters (125) of Kariskis bark
4 One kilogram (1 kg) of sugar cane juice with one hundred twenty five milliliters (125) of Kariskis bark and one hundred milliliters (100 ml) of mother vinegar
5 One kilogram (1 kg) of sugar cane juice with two hundred fifty milliliters (250) of Kariskis bark
6 One kilogram (1 kg) of sugar cane juice with one hundred twenty five milliliters (125) of Kariskis bark and two hundred fifty milliliters (250) of Kariskis bark

4. Evaluation of the products.
The researchers will utilize experimental and quantitative method for testing the bio enzymatic activity of Kariskis (Albizia Lebbekoides) in the fermentation of Sukang Iloko.
Experimental method will be used for testing the acidity of each treatment. The treatments and samples will be test for every two weeks using a digital Ph meter. While Quantitative method for testing the efficiency of the bio enzymatic activity of Kariskis (Albizia Lebbekoides) in the Sukang Iloko in terms of appearance, aroma and taste.
Furthermore, the mean will be use to find the efficiency and acceptability of Kariskis (Albizia Lebbekoides) bark as a fermentation agent.
Level of Efficiency and Acceptability of the Bioenzymatic Activity of Kariskis (Albizia Lebbekoides) bark in the fermentation of Sukang Iloko
Range of Mean Ratings Descriptive Interpretation
4.21-5.00 Very High
3.41- 4.20 High
2.61 – 3.40 Moderate
1.81 – 2.60a Low
1.00 – 1.80 Very Low

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