Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water kefir .... Experiment 2: Effect of different ratios of two sugar types on biomass growth an
INTRODUCTION
Problem statement
The COVID-19 pandemic has driven the need for alternative antiviral agents to reduce infection risk and improve patient recovery As health awareness rises, the demand for nutritious and functional foods has surged, with probiotic drinks emerging as a popular choice due to their digestive and immune system benefits These beverages are rich in beneficial microorganisms that aid in disease recovery and bolster immunity However, traditional probiotic drinks are often dairy-based, posing challenges for lactose-intolerant individuals Water kefir, made from a sugary solution, presents a viable alternative Scientific research supports the health-promoting effects of regular water kefir consumption, including anti-carcinogenic, antioxidant, anti-inflammatory, antifibrotic, and antimicrobial properties, leading to its growing global popularity.
Kefir drinks offer a variety of health benefits, but the relatively high alcohol content in current water kefir products poses a challenge for appealing to a wider audience across different age groups and regions This study aims to address this issue to expand the market potential for water kefir beverages.
Passion fruit is a nutritious tropical fruit packed with antioxidants, vitamins, and fiber that can enhance human health Gac fruit is particularly notable for its high levels of antioxidants and carotenoids, including lycopene and beta-carotene, which support various bodily functions Additionally, ginger is known for its potential anti-inflammatory, antibacterial, and antiviral properties The incorporation of these fruits and herbs into water kefir production results in a health-promoting beverage that aligns with consumer preferences for healthy hydration.
Research objectives
This thesis aimed to evaluate how different types and ratios of sugar influence the alcohol content in fermented kefir drinks The findings were utilized to recommend an effective production process for creating a low-alcohol kefir beverage infused with passion fruit, gac fruit, and ginger, catering to a broader customer base while preserving quality and nutritional value.
Research contents
The topic “Developing low alcoholic kefir beverage with passion fruit, gac fruit and ginger” included the following specific contents:
Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water kefir
Experiment 2: Effect of different ratios of two sugar types on biomass growth and alcoholic content of water kefir
Experiment 3: Effect of sugar concentration and kefir grains rate on biomass growth and alcohol content of water kefir
Experiment 4: Investigation of the appropriate fruit juice mixing ratio for kefir beverages and evaluate sensory of kefir beverages
Experiment 5: Determination the physiochemical and microbiological properties of the passion gac ginger kefir beverage
Experiment 6: Determination the shelflife of the passion gac ginger kefir beverage.
LITERATURE REVIEW
Water kefir
Water kefir is a vegan-friendly, probiotic-rich beverage made from sugar water and water kefir grains, which are not actual grains like rye or wheat These grains consist of a water-soluble polysaccharide gel called 'kefiran,' offering a mild, cord-like texture Water kefir grains vary in color from pale white to yellowish and can grow to the size of walnuts, resembling cauliflowers This makes water kefir an excellent alternative for those seeking probiotics and prebiotics without dairy.
Water kefir grains consist of a unique biofilm, which is a community of diverse yeast and beneficial bacterial strains coexisting in a stable culture These microorganisms adhere to a surface within a dextran matrix, a polysaccharide composed of sugars, lipids, and proteins, produced during the fermentation of sucrose.
The Symbiotic Culture of Bacteria and Yeasts (SCOBY) is a harmonious community of microorganisms that thrives on carbohydrate feeding, enabling the production of enzymes, carbon dioxide, alcohol (ethanol), and beneficial lactic acid that promotes probiotics Within each grain, various lactic acid bacteria coexist, with the Lactobacillus species being particularly dominant The microbial ecosystem of the grains and the resulting fermented beverage can differ based on fermentation conditions, such as time and temperature, as well as the substrates used.
Water kefir, traditionally produced on a small scale, is gaining popularity as a health-conscious beverage due to consumer lifestyle trends As its demand rises, there is an increasing need for a comprehensive understanding of its biology and production processes to ensure quality and consistency.
F1 water kefir is a fermented solution that develops over 48 hours at 25°C, featuring a diverse microflora and a mildly sweet, sour flavor This versatile solution can undergo a second fermentation to enhance its flavor and alcohol content, or it can be blended with fruit and vegetable juices for regular consumption.
Kefir beverages offer numerous health benefits, but the high alcohol content in current water kefir products limits their appeal to a broader audience To overcome this challenge and attract consumers of all ages, there is a need to develop low-alcohol kefir drinks, which would help expand the market for water kefir beverages.
2.1.2 Historical development of water kefir
In the late 19th century, early records revealed the use of water kefir grains in a fermented beverage made from the sweetened juice of tibicos, which grows on the pads of the nopal cactus in Mexico When reconstituted in a sugar and water solution, these grains formed hard granules that facilitated the spread of tibicos.
Speculations regarding the origins of water kefir have focused on regions such as the southern peninsula of Ukraine, the Caucasus mountains, and the northeastern Himalayas in Tibet, China The symbiotic relationship between active yeast and bacteria complicates the identification of its exact origin, as these microorganisms create various cultures for this effervescent drink Overall, water kefir cultures have been widely distributed globally and appear to have been utilized for thousands of years.
A reliable scientific article discusses the origins of Tibicos, highlighting a specific bacterium that shares properties with Tibicos This bacterium is known for producing dextran, akin to the polysaccharide matrix formed during the fermentation of sucrose by Lactobacillus hilgardii and Leuconostoc strains, which are the primary bacterial species found in water kefirs.
Water kefir grains, also known as sugar grains or tibicos in Mexico, are used to create the popular fermented drink tepache, made from piloncillo or brown sugar, pineapple skins, and cinnamon In France, these grains are called Graines Vivantes, while in Italy, they are referred to as Kefir di Frutta, and in Germany, they are known as Piltz.
Water kefir grains, also known as tibi, Japanese water crystals, or by various regional names such as African Bees, Aqua Gems, and Ginger Beer Plant, are widely recognized for their versatility Despite the different names and specific cultural compositions, the fundamental method of utilizing water kefir grains is consistent globally.
In recent years, the demand for probiotic products like yogurt, kefir, koumiss, and Yakult has surged globally Among these, kefir has gained significant popularity due to its high nutritional value and health benefits, making it a favored choice in Europe, Asia, and both North and South America.
2.1.3 Health benefits of water kefir beverage
Consumers today are increasingly drawn to products with functional characteristics that enhance their health The concept of "functional foods" has shaped perceptions of nutritious options Research has focused on kefir and kefir grains, aiming to deepen scientific understanding and explore the development of new, consumer-friendly nutritional products Regular consumption of kefir is associated with significant health benefits, including improved digestion, antioxidant activity, antibacterial effects, modulation of the gut microbiome, glucose control, weight management, cholesterol improvement, anti-inflammatory properties, and reduced cardiac and kidney hypertrophy Additionally, kefir exhibits strong antibacterial properties against foodborne pathogens and has been identified as a protective agent for the immune system against viral infections.
Boosting the immune system is one of the most effective strategies for preventing infections from various pathogens, including viruses Probiotic microflora in fermented milk drinks, such as kefir, provide health benefits through direct action of live cultures and their metabolites Kefir is particularly valuable due to its health-promoting and disease-prevention properties, which stem from its ability to enhance gut flora by increasing probiotic bacterial strains Regular consumption of kefir offers health benefits from probiotics like lactic acid, acetic acid, and antimicrobial proteins that exhibit bacteriostatic effects against numerous diseases The high concentration of bio-functional metabolites in kefir underscores the need for further research into its immune-boosting and antiviral properties Consequently, there is an increasing interest in conducting rigorous clinical trials to explore the effects of incorporating fermented functional drinks into diets or as dietary supplements.
2.1.4 Microorganisms and yeasts in water kefir
2.1.4.1 Representative microorganisms in water kefir
Research on the microbial diversity in water kefir has consistently shown the presence of various Lactobacillus species, although no single defining species has emerged Notably, Lactobacillus hilgardii and Lactobacillus nagelii play significant roles in water kefir grain communities due to their ability to produce exopolysaccharides (EPS) Additionally, Acetobacter sicerae, a distinct species of acetic acid bacteria (AAB), has recently been identified in water kefir.
Table 1 1 Overview of genera and species of yeasts and bacteria found in water kefir via culture- dependent and -independent methods
Horisberger (1969) [45] Lactobacillus brevis, Lactococcus lactis
Pidoux et al (1988) [23] Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides
Waldherr et al (2010) [7] Lactobacillus hilgardii
Acetobacter lovaniensis, Lactobacillus buchneri, Lactobacillus kefiri, Lactobacillus paracasei, Lactobacillus parabuchneri, Lactococcus lactis, Leuconostoc citreum
Acetobacter lovaniensis, Bacillus cereus, Gluconobacter liquefaciens, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus kefiri, Lactobacillus paracasei, Lactobacillus satsumensis, Lactobacillus sunkii
Gulitz et al (2011) [49] Acetobacter fabarum, Acetobacter orientalis, Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus hordei, Lactobacillus nagelii, Leuconostoc citreum, Leuconostoc mesenteroides
Hsieh et al (2012) [50] Lactobacillus hordei, Lactobacillus mali, Leuconostoc
Mesenteroides, Pseudomonas, Sporolactobacillus, Zymomonas mobilis
Gulitz et al (2013) [28] Acetobacter, Bifidobacterium, Clostridium,
Marsh et al (2013) [51] Acetobacter, Bifidobacterium, Gluconacetobacter,
Diosma et al (2014) [52] Pichia occidentalis, Saccharomyces cerevisiae
2.1.4.2 Representative yeasts in water kefir
Substrates and solvents for F1 water kefir fermentation
Sugar refers to a class of carbohydrates with the general formula (CH2O)n or Cn(H2O)n-1 for monosaccharides Commonly, it denotes sucrose, a crystalline sweetener widely used in foods and beverages Sucrose, a disaccharide, is found in most plants but is commercially extracted primarily from sugarcane and sugar beet It serves as the standard for sweetness, with other sweeteners being compared to it at 100%.
Cane sugar's chemical properties, including color, taste, sweetness, antioxidant capabilities, and reactions in aqueous solutions, significantly influence its everyday applications The color of cane sugar serves as an indicator of its purity, with lighter sugars representing the purest form of sucrose, containing minimal color and flavor molecules along with fewer organic and inorganic substances Additionally, brown sugar is commonly produced from cane sugar to enhance flavor.
Components White refined cane sugar Unrefined brown cane sugar
Cane sugar refining transforms raw cane sugar into highly pure white sugars, ensuring consistent quality and safety for home and food industry use The process effectively manages water, ash, and sugar content, allowing refined sugars to be stored for extended periods; even after 60 years at room temperature, they exhibit only slight color changes Refineries can optimize costs by returning low-grade products to manufacturers instead of processing them However, the refining process, along with the use of chemical compounds like sulfur dioxide and hydrogen peroxide for whitening, may influence the growth of microorganisms.
During the refining process, impurities are eliminated from all cane sugars Unrefined sweeteners, which retain most of the natural cane molasses, are the least processed This category includes traditional brown sugars like muscovado, panela, jaggery, and piloncillo, as well as those produced through more intricate drying methods, such as Sucanat.
Honey is primarily composed of sugars, which make up about 95% of its dry mass The main sugars in honey are monosaccharides, specifically fructose and glucose, resulting from the hydrolysis of sucrose Additionally, blossom honey contains oligosaccharides, including disaccharides like sucrose, maltose, turanose, and erlose, while honeydew honey features trisaccharides such as melezitose and raffinose.
Table 2 2 Chemical composition of honey [66]
Mineral compounds in honey vary in concentration from 0.02 to 1.03g/100g, with potassium being the most significant element, making up nearly one-third of the total minerals Research indicates that the concentration of trace elements in honey is influenced by its botanical origin, with lighter flower honeys containing lower mineral content compared to darker varieties like honeydew, chestnut, and heather.
Honey is a highly concentrated sugar solution with a high osmotic pressure, making
Bacillus species in honey can lead to significant issues for bees, although they pose no threat to humans To mitigate bee-related problems, it is essential to avoid discarding honey in open areas accessible to bees Additionally, honey contains naturally occurring osmotolerant yeasts that can cause undesirable fermentation, particularly in honeys with elevated moisture levels.
Grape molasses is rich in various sugars, including saccharose, glucose, fructose, maltose, and raffinose The average fructose content in grape molasses is approximately 28.42%, with values ranging from 22.34% to 34.69% In contrast, glucose levels vary from 27.57% to 41.11%, averaging around 31.67% A study by Simşek & Artık (2002) found that the fructose content in 25 commercially produced grape molasses samples ranged from 30.14% to 34.42%, while glucose levels varied from 30.73% to 34.99%.
Grapes are a rich source of nutrients and phenolic compounds, which enhance their antioxidant properties The macroelement content in grape molasses varies significantly among different grape varieties, ranging from 48.70 mg/kg to 5109.56 mg/kg.
Water is the primary component in soft drinks in general, most experiments used bottled Lavie water
Table 2 3 The of composition of Lavie natural mineral water ( Lavie product’s label)
Figure 2 5 The chemical formula of sodium bicarbonate
Sodium bicarbonate, commonly referred to as baking soda, is a white crystalline solid with the chemical formula NaHCO3, typically found in a fine powder form The concentration of bicarbonate ions in the water significantly influences the pH, development of water kefir grains, diversity of microbial species, and the synthesis of metabolites during water kefir fermentation.
Sodium bicarbonate, or baking soda (NaHCO3), is a white crystalline solid commonly found as a fine powder The concentration of bicarbonate ions in water used for water kefir fermentation plays a crucial role in influencing pH, grain formation, microbial diversity, and metabolite production A high bicarbonate buffer capacity enhances the growth and metabolism of lactic acid bacteria (LAB) compared to yeasts, leading to a favorable LAB to yeast ratio and higher lactic acid to ethanol concentration ratios Conversely, inadequate water buffer capacity significantly hampers the growth of water kefir grains.
Passion fruit
Chemical composition of passion fruit
Figure 2 6 Passion fruit (Passiflora edulis Sims)
Passion fruit (Passiflora edulis Sims) is a tropical and subtropical fruit celebrated for its delicious flavor and impressive nutritional benefits Traditionally, it has been utilized in folk medicine to address various health issues, including anxiety, asthma, urinary tract infections, and diabetes.
Table 2 4 Composition of passion fruit [76] Nutritional value per 100 g (3.5 oz)
Application of passion fruit in fermented foods
The addition of passion fruit juice significantly boosts the polyphenol content and antioxidant activity of set yogurt This fruit is known for its antibacterial properties and is a key ingredient in coalho cheese, a traditional Brazilian delicacy Moreover, passion fruit enhances the nutritional value and flavor of various fermented beverages, including wine and vinegar Recent studies have also explored its application in the production of water kefir.
Gac fruit
Chemical composition of gac fruit
Gac fruit (Momordica cochinchinensis Spreng.), a member of the Cucurbitaceae family first identified in Vietnam, is recognized for its exceptionally high levels of lycopene and beta-carotene in its oily seed membrane, making it one of the richest natural sources of carotenoids This fruit has been valued for its health benefits and has a long history of use in food and traditional medicine throughout Southeast Asia, with various components such as seeds, oil, and roots being utilized in traditional healing practices.
Table 2 5 Composition of 100g gac fruit aril [86]
Gac fruit, often referred to as a "super fruit" or "heaven's fruit," is rich in carotenoids, phenolics, and vitamin C, offering significant health benefits such as anti-inflammatory and antioxidant effects, cancer prevention, vision maintenance, and immune modulation Its high phytonutrient content across all parts—aril, seeds, pulp, and peel—enhances its therapeutic and pharmacological properties Notably, the aril of gac fruit contains the highest total phenolic content, making gac fruit powder a valuable resource for nutritional supplements, natural colorants, and therapeutic applications.
Application of gac fruit in fermented foods
Research on the use of gac fruit in fermented foods is limited, with only one study focusing on lactic acid bacteria fermenting a blend of gac juice However, gac fruit shows promise for incorporation into various products, such as pasteurized juice, milk drinks, sticky rice, pasta, sauces, and fermented items like yogurt, kefir, and cider.
Ginger
Ginger (Zingiber officinale), a member of the Zingiberaceae family, is a widely used spice, particularly in Asian cuisine It contains various bioactive components, including phenolic and terpene compounds The primary terpene components identified in ginger are sesquiterpene hydrocarbons and phenolic compounds like gingerol and shogaol Additionally, lipophilic rhizome extracts yield active gingerols that can be transformed into shogaols, zingerone, and paradol Ginger is also rich in amino acids, dietary fiber, ash, protein, phytosterols, vitamins (such as nicotinic acid and vitamin A), and essential minerals.
Figure 2 8 Chemical structure of active ingredients of Ginger [96]
Figure 2 9 The main properties of ginger [97]
Application of ginger in fermented foods
Natural ginger extract enhances beer by adding bioactive phytoconstituents and essential nutrients like proteins, vitamins, and minerals, which support consumer health Additionally, it is widely utilized in the production of kombucha and kefir beverages.
Pectinase
Pectinases, or pectinolytic enzymes, are a group of enzymes that break down pectic compounds These enzymes play a crucial role in various applications, such as processing plant fibers, fermenting tea and coffee, and treating industrial wastewater.
Application of pectinase in fruit juice treatment
Pectinases play a crucial role in the food processing industry by enhancing juice clarification, improving color and yield, and treating fruit mash These enzymes effectively break down pectin in fruits and disintegrate cell walls, resulting in significantly higher enzyme yields for each ton of fruit processed.
MATERIALS AND METHODS
Materials
3.1.1 Production of F1 water kefir beverage
The activated water kefir grains were obtained from Nurturing Foods Co., Ltd (Vietnam)
Grape molasses and floral honey were sourced from Le Nhan Grape Syrup in Ninh Thuan, Vietnam, and Tracybee in Vietnam, respectively Additionally, white sugar was obtained from Thanh Thanh Cong - Bien Hoa JSC in Vietnam, while organic brown cane sugar was supplied by Wangkanai in Thailand.
Pectinase enzyme was purchased from ICFood Co., Ltd (Vietnam)
Figure 3 1 Activated water kefir grains (Nurturing Foods Co., Ltd, Vietnam)
Figure 3 2 Types of sugars used in water kefir fermentation Procedure
A mixture of sugars and NaHCO3 was combined with 240 mL of potable water to create a homogeneous solution This sugar medium was pasteurized at 60 °C for 30 minutes to reduce microorganisms, followed by cooling to room temperature in preparation for inoculating the fermentation culture.
Activated water kefir grains were added into 240 mL of the medium and the fermentation process was conducted at 25 o C for 48 hours in a laboratory
After the fermentation process, the fermentation batch is filtered to get the F1 solution for the juice mixing, and the kefir grains are reused for other fermentation batches
Figure 3 3 Production of F1 water kefir beverage [8], [27]
3.1.2 Preparation of passion gac ginger juice
Passion fruit and ginger were obtained from Coopmart (Ho Chi Minh City, VietNam) Gac powder was purchased from Gavi Moocos Co., Ltd (Vietnam)
Figure 3 4 Production of passion fruit juice
Passion fruits were meticulously selected to ensure only undamaged and ripe fruits were used After washing to eliminate impurities, the fruits were cut to extract the pulp The pulp was then strained to remove seeds, and 0.1% pectinase was added to the juice for enzyme treatment The mixture was incubated at a temperature of 45-55 °C with an agitation rate of 150 rpm To inactivate the enzymes, the juice was pasteurized at 90 °C for 5 minutes in a hot water bath before being combined with other ingredients.
Figure 3 5 Production of gac fruit solution
To develop a uniform solution, gac fruit powder was fully dissolved in hot water at different ratios These solutions were subsequently mixed with additional ingredients to establish the juice ratio for the subsequent experiment.
Figure 3 6 Production of ginger juice
Ginger is first thoroughly washed to eliminate physical impurities, followed by the careful removal of its skin with a sterilized knife Any damaged or spoiled sections are discarded to maintain high quality for ginger extraction Finally, the ginger juice is extracted using a juice presser and mixed with other ingredients.
Figure 3 7 Production of passion gac ginger juice
The passion fruit juice, gac fruit solution, and ginger juice were combined and homogenized to create a uniform mixture This fruit juice blend was then pasteurized at 90°C for 5 minutes to eliminate harmful microorganisms and deactivate enzymes After pasteurization, the juice was cooled to room temperature before being combined with F1 water kefir solution.
3.1.3 Production of passion gac ginger kefir beverage
The passion gac ginger water kefir beverage was created by blending F1 water kefir with passion gac ginger juice in varying ratios of 10%, 20%, and 30% Following the mixing process, the beverage was bottled and stored in a refrigerator at temperatures between 3 to 6 degrees Celsius to assess the storage conditions of the water kefir drinks Sensory evaluations were conducted to determine the quality of the final product.
Research contents
3.2.1 Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water kefir
The experiment aims to assess how various types of sugar influence both the alcohol production and the growth of water kefir grains The goal is to identify the optimal sugars that minimize alcohol content while enhancing the growth of the grains.
In a study, various sugars were analyzed, including 17g of refined white cane sugar, 17g of brown cane sugar, 20g of honey, and 22g of grape molasses, combined with 0.25g of NaHCO3 and 240 ml of Lavie water to achieve a mixture of 7.0 ± 0.5 o Brix The pH of the resulting medium was measured at 8.20 ± 0.05 This sugar medium was then homogenized, pasteurized at 60 o C for 30 minutes, and allowed to cool to room temperature Subsequently, 36 g of activated water kefir grains were introduced to the 240 mL medium, and fermentation was carried out at 25 o C for 48 hours in a laboratory setting.
After 48 hours of fermentation, the batch is filtered to produce the F1 solution, which is then analyzed for alcoholic content, pH, and total soluble solids The remaining kefir grain residue is utilized for biomass determination.
3.2.2 Experiment 2: Effect of different ratios of two sugar types on biomass growth and alcoholic content of water kefir
The experiment aims to assess how different sugar ratios of the two most effective sugars identified in previous experiments influence both the alcohol content and the growth of water kefir grains This will help in identifying the optimal sugar ratio for fermenting a low-alcohol water kefir beverage.
The mass of two optimal sugars with mixing ratios: 1:1; 1:3; and 3:1 were examined in this experiment
A mixture of sugar and 0.25g NaHCO3 was combined with 240 ml of Lavie water to achieve a concentration of 7.0 ± 0.5 o Brix, with a recorded pH of 8.20 ± 0.05 The sugar medium was then homogenized, pasteurized at 60 o C for 30 minutes, and subsequently cooled to room temperature.
36 g of activated water kefir grains were added to 240 mL of the medium and the fermentation process was conducted at 25 o C for 48 hours in a laboratory [8]
After 48 hours of fermentation, the batch is filtered to obtain the F1 solution, which is then analyzed for alcoholic content, pH, and total soluble solids The remaining kefir grain residue is utilized for biomass determination.
3.2.3 Experiment 3: Effect of sugar concentration and kefir grains rate on biomass growth and alcohol content of water kefir
Experiment 3.1: Effect of sugar concentration on biomass and alcohol content of kefir water
The experiment aims to assess how varying sugar concentrations affect the growth of water kefir grains and the resulting alcohol content By identifying the optimal sugar concentration, the study seeks to enhance the fermentation process for low-alcohol water kefir beverages.
This experiment focused on three concentrations of the optimal sugar ratio (5%-12g, 10% -17g, and 15% -24g)
A mixture of sugar and 0.25g NaHCO3 was combined with 240 ml of Lavie water and homogenized The medium was pasteurized at 60 °C for 30 minutes and then cooled to room temperature Following this, 36 g of activated water kefir grains were introduced to the medium, and fermentation occurred at 25 °C for 48 hours After fermentation, the mixture was filtered to obtain the F1 solution for analysis of alcoholic content, pH, and total soluble solids, while the kefir grain residue was used for biomass determination.
Experiment 3.2: Effect of kefir grain rate on biomass and alcohol content of kefir water
The experiment aims to assess how varying percentages of kefir grains influence the growth of water kefir grains and the alcohol content in low alcoholic water kefir beverages The goal is to identify the optimal kefir grain rate for effective fermentation.
The optimal sugar concentration and 0.25g NaHCO3 [27] were blended with 240 ml of Lavie water The sugar medium is homogenized, pasteurized at 60 o C for 30 minutes [8], cooled down at room temperature
Three rates of kefir grains (5 %-12g, 10% -36g, 15% -48g) were added.to 240 mL of the medium and the fermentation process was conducted at 25 o C for 48 hours in a laboratory
After 48 hours of fermentation, the batch is filtered to obtain the F1 solution, which is then analyzed for alcoholic content, pH, and total soluble solids The remaining kefir grain residue is utilized for biomass determination.
3.2.4 Experiment 4: Investigation of the appropriate fruit juice mixing ratio for kefir beverages and evaluate sensory of kefir beverages
3.2.4.1.Investigation of gac fruit powder portions
Our kefir drink is primarily flavored with passion fruit juice, known for its appealing taste As a result, this juice constitutes the largest percentage of ingredients in our mixed juice.
The study explores the combination of gac fruit powder and passion fruit juice, selected for their high nutritional content, including vitamins, minerals, and antioxidants that benefit human health However, the undesirable flavor of gac fruit, characterized by an oily smell and irritating taste, necessitates an investigation to identify the optimal amount of gac fruit powder to enhance the beverage's value while improving the flavor profile of the final kefir drink.
The study examined gac fruit powder at three different concentrations: 0.5%, 1.0%, and 1.5% The gac fruit powder was fully dissolved in hot water to decrease total soluble solids, which is detrimental to low alcoholic kefir production, while also lowering the acidity of the final product This red solution was then combined with 100g of enzyme-treated and heated passion fruit juice, with the ingredient ratios detailed in the accompanying table.
Table 3 1 Formula of gac fruit powder ratios in the fruit juice
Sample Gac fruit powder Passion fruit juice Potable water
This fruit juice (10%) was mixed with the F1 water kefir solution (90%), and the beverage was
28 up with the most favorable sample Therefore, the appropriate amount of gac fruit powder added was concluded
Figure 3 9 Kefir beverages made from passion fruit juice and gac fruit powder 0.5%; 1.0%;
3.2.4.2.Investigation of ginger portions to minimize the off-odor
The kefir drink made from the two selected ingredients boasts a pleasant flavor profile, featuring a mild sourness and slight sweetness characteristic of traditional kefir However, the beverage's aroma was less appealing, prompting the addition of ginger to address this issue This experiment aims to identify the optimal ginger ratio for enhancing the final product.
Ginger, a highly regarded aromatic root vegetable, is celebrated for its numerous health benefits Research has explored the incorporation of ginger into kefir beverages, highlighting its appealing aroma, which can enhance the overall sensory experience.
Analysis methods
3.3.1 Determination of the alcohol content of kefir water after each fermentation
An alcoholic beverage analysis system utilizes infrared spectroscopy techniques to analyze molecular infrared spectra The Alcolyzer Plus Beer from Anton Paar GmbH, Graz, Austria, was used to directly measure the alcohol content at Nurturing Foods Co., Ltd in Vietnam Calibration of the equipment was conducted using distilled water, and a 15ml sample was filtered through cotton wool before being pumped into the device via a filling unit The results were expressed as percentage volume per volume (% v/v).
3.3.2 Determination of the biomass of water kefir grains
After 48 hours, at the end of the first fermentation process, the changes in the biomasses of water kefir grains in the water kefir drinks were gravimetrically evaluated [105] After fermentation, grains were sorted aseptically using stainless steel sieves (5 cm) Grains were rinsed with sterile distilled water before being placed on autoclaved sterile aluminum weighing plates using sterilized paper towels to remove excess water After removing the paper towels, the water kefir grains were weighed with an analytical balance Biomass was calculated as a percentage of the difference between final and initial grain weights
Lactobacillus spp counts were evaluated using a De Man, Rogosa, and Sharpe (MRS) medium
(Merck, Darmstadt, Germany) after 48 hours of incubation at 37 o C with 5% CO2 The counts of
Lactococcus spp and Streptococcus spp were plated on M17 medium (Merck, Darmstadt,
Germany) and cultured for 48 hours at 37 o C in 5% CO2
Yeasts were cultivated for 5 days at 25 o C on Potato Dextrose Agar (PDA) medium (Merck, Darmstadt, Germany) with 0.14% additional lactic acid [109]
The pH meter (PH818 SmartSensor, China) is utilized for pH testing, requiring the test sample temperature to be adjusted to approximately 25°C Additionally, the temperature compensator control should be set to the recorded temperature, in accordance with AOAC 981.12 standards.
3.3.5.Determination of total soluble solids (TSS)
Total soluble solids (TSS) were determined using a hand digital refractometer (Master - M Atago Co., Ltd., Tokyo, Japan) and recorded as o Brix using the AOAC method 932.14
A sensory evaluation was performed with 15 trained panelists who were familiar with kefir's qualities They underwent orientation sessions using untreated kefir samples and scored each sample on a 9-point hedonic scale, ranging from 1 (severely hated) to 9 (strongly liked) The sensory data was recorded using a specific assessment form, which can be found in Appendix B.
All experiments were conducted in triplicate, and the results are presented as mean ± standard deviation of the mean The data were analyzed using a one-way analysis of variance (ANOVA) with SPSS 18.0, and a P-value of less than 0.05 was deemed statistically significant.
RESULTS AND DISCUSSION
Effect of different sugar types on biomass growth and alcoholic content of water
Table 4 1 Results effect of sugar types on kefir water growth
Sample Biomass growth (%) Total soluble solids ( o Brix) pH % alcohol RWK 6.23 ± 0.34 a 5.13 ± 0.11 a 5.85 ± 0.02 a 1.39 ± 0.01 a
*RWK: white refined sugar water kefir; BWK: brown cane sugar water kefir; GMK: grape molasses water kefir; HWK: honey water kefir Values with different lowercase letters means differ significantly (p0.05) and an increased alcohol content of 1.5% (P