Research on fermented beverage by using the residue of (angelica sinensis (oliv ) diels) root

DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Thesis Title: Research on fermented beverage b

UNIVERSITY OF AGRICULTURAL AND FORESTRY

CAO HONG LE

RESEARCH ON FERMENTED BEVERAGE DEVELOPMENT FROM

THE RESIDUE OF (Angelica sinensis (Oliv.) Diels) ROOT

BACHELOR THESIS

Study Mode : Full time Major : Food Technology Faculty : Advanced Education Program Office Batch : 2017 - 2021

Thai Nguyen, 2022

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program: Bachelor of Food Science and Technology

Thesis Title: Research on fermented beverage by using the residue

of (Angelica sinensis (Oliv.) Diels) root

Supervisor's Signature:

This study aimed to investigate the effects of some factors on the fermentation

process of the residue of (Angelica sinensis (Oliv.) Diels) root by using

Vietnam in addition this process will help to use by-products of the instant tea

production process from (Angelica sinensis (Oliv.) Diels) In this research,

years of cultivation The root was extracted by 70% (v/v) ethanol and dried to

obtain a residue, subsequently fermented with Saccharomyces bayanus The

fermentation process was researched and the most suitable parameters were determined The result gives information that the material/distilled water ratio was 1/30, fermentation at pH=4, initial concentration of sugar was 23°Brix, the added yeast ratio was 3% (v/w) with the time for fermentation was 8 days, the alcohol content of final product was 9.5% (v/v) and the chemical, physical, sensory, and microbiological characteristics of final product satisfied the requirements of the Vietnam Standard for fermented beverages

Saccharomyces bayanus, temperature, pH, °Brix, alcohol content, (material/distilled water) ratio, fermentation parameters

Number of pages: 70

Date of submission June 1st, 2022

ACKNOWLEDGEMENT

This experiment was carried out at the Department of Food Technology, Faculty of Biotechnology and Food Technology, at Thai Nguyen University of Agriculture and Forestry in the year 2021

First and foremost, I would like to thank my supervisor MSc.Dinh Thi Kim Hoa and MSc.Vi Dai Lam , for helping me to overcome the obstacles during the period under the thesis They were always willing to assist me whenever I needed

it and directed me in the right direction for finishing the project

Secondly, I would like to thank Dr Nguyen Tien Cuong, at Ha Noi University of Science and Technology as well as MSc Luu Hong Son a member

of the Department of Food Technology, Faculty of Biotechnology and Food Technology, for providing me with a wealth of information and professional advice in my thesis

Thirdly, I would want to convey my heartfelt appreciation to my family and friends, those who have always been there for me, supporting me whenever I've run into a roadblock and assisting me in getting through the tough period of finishing this bachelor thesis

Finally, this thesis cannot escape flaws due to a lack of knowledge and practice time I'm looking forward to getting feedback from professors and friends

on how to enhance my thesis

TABLE OF CONTENT

ACKNOWLEDGEMENT iii

TABLE OF CONTENT iv

LIST OF FIGURES 1

LIST OF TABLE 2

LIST OF ABBREVIATIONS 3

CHAPTER I INTRODUCTION 4

1.1 Research rationale 4

1.2 Research’s objective 5

1.2.1 Overall objectives 5

1.2.2 Detail objectives 5

1.3 Research question 5

1.4 Limitation 5

CHAPTER II LITERATURE REVIEW 6

2.1 Overview about Angelica sinensis (Oliv.) Diels 6

2.1.1 Characteristics of Angelica sinensis (Oliv.) Diels 6

2.1.2 Overview of the chemical compositions of Angelica sinensis (Oliv.) Diels 7 2.1.3 Overview of biological effects (pharmacology) of Angelica sinensis (Oliv.) Diels 9

2.2 Some kind of products in the current market 10

2.3 Fermentation technology 11

2.3.1 Types of Fermentation Processes 12

2.4 Mechanism of alcoholic fermentation 12

2.5 Fermented beverage 15

2.5.1 Definition 15

2.5.2 Historical development of fermented beverage 16

2.5.3 Classification of fermented beverages 17

2.5.4 Health benefits of fermented beverages 18

2.6 Low alcohol beverage 19

2.6.1 The method used in the low-alcohol production 20

2.7 Saccharomyces bayanus 22

2.7.1 Morphology 22

2.7.2 Structural 22

2.7.3 Chemical components 23

2.8 Microbiology in fermented beverages 23

2.8.1 Yeast 23

2.8.2 Lactic acid bacteria 24

2.9 Some factors affect to fermented beverage 27

2.9.1 Influence of temperature 27

2.9.2 Influence of oxygen on fermentation 28

2.9.3 Influence of pH 29

2.9.4 Influence of sugar contrentration 29

2.9.5 Influence of yeast ratio on fermentation medium 30

2.9.6 Influence of alcohol content 30

2.9.7 Influence of light 31

2.9.8 Influence of organic acids 31

2.9.9 Influence of minerals 31

2.10 By-products of fermentation 31

2.10.1 Formation of organic acids 31

2.10.2 Glycerol 32

CHAPTER III MATERIALS, RESEARCH CONTENTS AND METHODOLOGY 33

3.1 Material and research scope 33

3.1.1 Material 33

3.1.2 Research scope 33

3.2 Work place and time to proceed 33

3.3 Chemicals, equipment 33

3.4 Research content 35

3.5 Research methods 37

3.5.1 Experimental design method 37

3.5.2 The biochemistry method 41

3.6 Data satistical analysis methods 50

CHAPTER IV RESULT AND DICUSSION 51

4.1 Result for determining the component of raw materials 51

4.2 The result of studying some factors affecting on the quality of product 52

4.2.1 The result of effecting material/ distilled water ratio on the quality of product 52

4.2.2 The result of the effect of the pH on the quality of product 53

4.2.3 The result of the research on initial sugar concentration affecting the quality of product 55

4.2.4 The result of research on yeast ratio affects to the quality of product 57

4.2.5 The result of research on fermentation time affects the quality of the product 58

4.3 Completing the process of producing fermented beverage from the residue of Agelica sinensis (Oliv.) Diels root 61

4.4 Evaluation the quality of final product and prelimary cost estimatation final product 62

4.4.1 Evaluation the quality of final product 62

4.4.2 Prelimary cost estimation final product 63

CHAPTER V CONCLUSION AND RECOMMENDATION 64

5.1 Conclusion 64

5.2 Recommendation 64

CHAPTER VI REFERENCE 65

CHAPTER VII APPENDIX 70

7.1 Appendix ballot for product sensory assessment score 70

7.2 Appendix statistical analysis 73

7.2 Appendix picture 84

LIST OF FIGURES

Figure 2.1 Angelica sinensis (Oliv.) Diels 6

Figure 2.2 Ferulic acid 8

Figure 2.3 (Dong Quai Root, 当归, Radix Angelica sinensis (Oliv.) Diels powder) 11

Figure 2.4 Angelica sinensis (Oliv.) Diels, Dried Root Liquid Extract 11

Figure 3.1 The processing of produce fermented beverage from the residue of Angelica sinensis ( Oliv.) Diels root according to (Wyman, C.E., 1996.) 35

Figure 3.2 Reducing sugar and DNS color responses 46

Figure 3.3 Processing was depicted by using the distillation method 47

Figure 4.1 Result for determining the component of raw materials 51

Figure 4.2 Process of making a fermented beverage from the residue of (Angelica sinensis (Oliv.) Diels root 61

LIST OF TABLE

Table 2.1 Several microbes play a significant role in fermented beverage (Yamada

& Sgarbieri, 2005) 26Table 3.1 Experiment chemicals 33Table 3.2 Laboratory instruments 34Table 3.3 Experimental design to determine the effect of material/ distilled water

ratio on the quality of product 37Table 3.4 Experimental design to determine the effect of pH on the quality of product 38Table 3.5 Experimental design to determine the initial sugar concentration affect

on the quality of product 39Table 3.6 Experimental design to determine the effect of yeast ratio on the

fermentation process 40Table 3.7 Experimental design to determine the effect of fermentation time on the

quality of product 41Table 3.8 Fermented beverage weight coefficient for sensory evaluation 49Table 3.9 Score of quality levels 50Table 4.1 Affecting of material/ distilled water ratio on the quality of product 52Table 4.2 Affecting of material/ distilled water ratio on the sensory quality of product 52Table 4.3 Affecting of pH on the quality of product 53Table 4.4 Affecting of pH on the sensory quality of product 54Table 4.5 Affecting of initial sugar concentration on the quality of product 55Table 4.6 Affecting of initial sugar concentration on the sensory quality of product 56Table 4.7 Affecting of yeast ratio on the quality of product 57Table 4.8 Affecting of yeast ratio on the sensory quality of product 58Table 4.9 Affecting of fermentation time on the quality of product 59Table 4.10 Affecting of fermentation time on the sensory quality of product 59Table 4.11 Result of the product's quality analysis 62Table 4.12 Estimation costs for 1 liter of fermented beverage from the residue of

Angelica sinensis (Oliv.) Diels) root 63

LIST OF ABBREVIATIONS

CHAPTER I INTRODUCTION 1.1 Research rationale

endemic to China The root of AS which is known in Chinese as Danggui has been

cultivated and used in China for more than 2000 years Many years ago, there is a

variety of research found out that the root of Angelica sinensis (Oliv.) Diels include

ferulic acid, Z-ligustilide, butylidenephthalide, and various polysaccharides These compounds are used for tonifying blood, treatment of anemia, rheumatism, and menstrual problems (Chao & Lin, 2011) After extracting, the majority of

valuable products A considerable amount of ginsenosides or polysaccharides still

is kept in residue Due to this reason the residue of Angelica sinensis (Oliv.) Diels)

root has a significant advantage to generate valuable products by reducing the lignin, cellulose and allows yeast to participate in the conversion of glucose into ethanol during fermentation according to (Wyman,1996) In this day and age, the varieties of functional foods that are available include products such as dairy foods, confectionery, baked goods and cereals, meat products, fermented beverages, and spreads Among them, fermented beverages have been proved to be the most active functional food category due to it processing several benefits such as the possibility

to meet consumer demand, convenience, size, shape, and appearance, better storage options for refrigerated and self-stable products with ease of distribution Fermented beverage form a key part of the food industry which are intended for human consumption Indeed that in Vietnam as well as in the world, the trend of using fermented beverage from medicinal herbal is increasing As a result, some technical methods or procedure is expected to develop the new fermented beverage product from medicinal herbal and also simultaneously encourage the long-term development of fermented beverages Low raw material and operation costs are one of the priorities in the food industry production As a consequence, the current tendency is to use low-cost raw resources such as agricultural by-products This has encouraged researchers to seek out new raw material sources and make use of copious waste resources in order to form a better product Based on the discussion

above, I have conducted the project " Research on fermented beverage

development from the residue of Angelica sinensis (Oliv.) root "

1.2 Research’s objective

1.2.1 Overall objectives

The overall objective of this research is to enhance the utility of the Angelica

high sensory value that is compatible with the requirements of consumers and also

aims to mitigate the by-products in the laboratory

1.2.2 Detail objectives

- Evaluating the raw material component of Angelica sinensis (Oliv.) Diels root

- Using the residue of Angelica sinensis (Oliv.) Diels root after being

extracted with alcohol to create the fermented beverage product

- Investigating the potential factors that could influence to fermentation

process by using the residue of Angelica sinensis (Oliv.) Diels root after being

extracted with alcohol

- Completing the process of producing fermented beverages from the residue

of Angelica sinensis (Oliv.) Diels root and evaluating the quality of final product

CHAPTER II LITERATURE REVIEW 2.1 Overview about Angelica sinensis (Oliv.) Diels

2.1.1 Characteristics of Angelica sinensis (Oliv.) Diels

China also known as Danggui in Chinese which is a perennial herb found in

mainland China, Taiwan, Japan, and Korea Other common names for AS include Chinese Angelica, dang gui (Chinese), toki (Japanese), tanggwi (Korean), and

kinesisk kvan (Danish) Angelica is a member of the Umbelliferae family that blooms in white umbrella-like clusters from June to July Angelica sinensis (Oliv.)

Diels can be divided into three distinct parts, namely head, body, and tail, which are reported to have different therapeutic effects The head is mainly used to stop bleeding, the body to nourish the blood and the tail to quicken the blood A normal plant reaches a height of about six feet (two meters) The medicinal benefits of the dried root are highly valued It has a peculiar flavor that is a blend of bitter, sweet,

and pungent Chinese herbalists have used Angelica for thousands of years to

strengthen heart, lung, and liver meridians, as well as lubricate the bowel It is considered a blood tonic, and has been used by generations of women for health concerns such as menstrual pain and regulating the menstrual cycle (Ross, 2001)

Figure 2.1 Angelica sinensis (Oliv.) Diels

In recently Angelica sinensis (Oliv.) Diels has been found in Vietnam since

1960 The majority of Angelica sinensis (Oliv.) Diels is grown in some provinces,

for example: Lao Cai, Hoa Binh, Lai Chau, Da Lat, Lam Dong A typical plant grows to approximately 40 to 80cm, generally growing in the cool humid temperature and is located between 2,000 and 3,000 meters above sea level

abbreviated or stemless stalks Angelica sinensis (Oliv.) Diels, according to

traditional chinese medicine, is a really common herb in oriental medicine that helps to make a cure for women and is also indicated in many prescriptions as tonics and to treat diseases such as, anemia-related headaches, weakness, cardiovascular disease, back pain, chest pains, arthritis, pain in the limbs, paralysis, constipation, itchy sores, hemophilia, irregular menstruation, dysmenor (taken 7

days before menstruation) (Giacomelli et al., 2017) Angelica sinensis (Oliv.) Diels

cures irregular menstruation, blood stasis, amenorrhea, uterine prolapse, bleeding, rheumatism, poisonous constipation, anemia, infectious diseases, high blood pressure, and used as a pain reliever, anticonvulsant, appetite stimulant by drinking

10 - 20g per day in the form of a decoction or Women who drink a decoction of

it helps to ease labor and reduce the pain

According to recent medical research Angelica sinensis (Oliv.) Diels includes

numerous beneficial active compounds, namely essential oils, the most vital active compounds is Z-ligustilide, which stimulates blood circulation, and n-butylphtalid, which is used to stymie ischemic stroke, immune-boosting and tumor-suppressing polysaccharides; coumarins have hemodynamic effects; phytoestrogens control uterine contractions, reduce oxytocin-type effects of pituitary hormones, are anti-inflammatory, and lower blood pressure; ferulic acid inhibits platelet aggregation (Chao & Lin, 2011)

2.1.2 Overview of the chemical compositions of Angelica sinensis (Oliv.) Diels

Several publications on the chemical compositions of Angelica sinensis

(Oliv.) Diels have been published in recent years, notably in China, comprising many on the chemical composition of the root and some on the stem and leaves of

this species Angelica sinensis (Oliv.) Diels has been used in Chinese medicine to treat menstrual disorders According to the research of Lin et al 1998 showed that

over 70 compounds have been isolated and identified from AS, encompassing essential oils such as ligustilide, butylphthalide and senkyunolide, phthalide dimers, organic acids and their esters such as ferulic acid, coniferyl ferulate,

polyacetylenes, vitamins and amino acids Z-ligustilide is the major lipophilic component of essential oil constituents and a characteristic phthalide component

of a variety of Umbelliferae plants, according to Naito T et al 1996 Z-ligustilide has a wide range of pharmacological properties, including anticancer, anti-inflammatory, anti-oxidant as well as neuroprotective activities and so on (Chao

& Lin, 2011)

Phthalides

Monomeric phthalides, such as Z-ligustilide and phthalide dimers, are found

in phthalaides According to author Lin LZ.et al.1998 the majority of phthalides identified is relatively non-polar, the fraction of which can be extracted with solvents such as hexanes, pentane, petroleum ether, methanol, 70% ethanol and dichloromethane Z-ligustilide has a wide range of pharmacological properties, including anticancer, antiinflammatory, anti-oxidant as well as neuroprotective activities and so on (Chao & Lin, 2011)

Organic acids

Many organic acids are found in Angelica sinensis (Oliv.) Diels For example,

ferulic acid extracted from AS is commonly utilized as a quality indicator chemical

for AS and its products According to (Chao WW.et al 2009), ferulic acid was

extracted from AS by using ethyl acetate Ferulic acid is an antioxidant, inflammatory, and anti-cancer agent and apart from its effects against Alzheimer’s disease, it possesses anti-hyperlipidemic, antimicrobial, and anti-carcinogenic properties

anti-Figure 2.2 Ferulic acid

Polysaccharides

Fructose, galactose, glucose, arabinose, rhamnose, and xylose are among the polysaccharides found in AS, according to (Wang QJ et.al 2003) The efficacy of

which are extracted with water as the initial extraction solvent In recently many researchers have been interested in polysaccharides because it brings many benefits for our health for example anti-tumor and immunomodulatory effects

2.1.3 Overview of biological effects (pharmacology) of Angelica sinensis (Oliv.)

Diels

to its diverse components Such characteristics include Anticoagulant activity, Immune Support and Hematopoiesis, Anti-inflammatory effects, Anti-cancer effects, Anti-cardiovascular effects, Immunomodulatory effect, Neuroprotective effects, Anti-oxidative activities, Anti-hepatotoxic effects as well as Kidney protective effects

Anti-inflammatory effects: Ferulic acid and Z-ligustilide, two major

compounds in AS may contribute to the anti-inflammatory activity of AS According to (Chao WW et al, 2010) revealed that the ethyl acetate extract of AS inhibits the production of inflammatory mediators thereby alleviating acute inflammatory hazards

Anti-cancer effects The three main AS phthalides, namely

n-butylidenephthalide, senkyunolide A and Z-ligustilide, decrease cell viability of colon cancer HT-29 cells dose- dependently.Yu et al reported that pretreatment of the PC12 cells with Z-ligustilide attenuates H2O2- induced cell death, attenuates an increase in intracellular reactive oxygen species (ROS) level, decreases Bax expression and cleaves caspase-3 and cytochrome C (Yu Y, et al 2008)

Anti-cardiovascular effects: (Gua AJ, et al 2009) demonstrated that excess

adipose tissue can lead to insulin resistance and increases the risk of type II diabetes and cardiovascular diseases Water and 95% ethanol extracts of AS effectively decrease fat accumulation in adipo cytes and reduce triglyceride content Yeh JC, et al illustrated that n-butylidenephthalide is anti-angio- genic and

is associated with the activation of the signaling pathways

Antiplatelet effect: Ferulic acid in AS, according to (Zhu DP 1987), can decrease the polymerization of platelets in blood circulation It inhibits the release

of 5-hydroxytryptamine (5-HT) and adenosine diphosphate (ADP) by platelets (ADP) Platelet aggregation and serotonin release were observed to be inhibited

by both ferulic acid and an aqueous extract of AS

Neuroprotective effects: AS extract protects Neuro 2A cells against b-amyloid

(Ab) peptide-induced oxidative damage via ROS, MDA, and glutathione (GSH) and restores mitochondrial transmembrane potential levels, according to Huang et al Z-ligustilide suppresses the TNF-a-activated NF- B signaling pathway, which may help explain why it protects rats against Ab peptide-induced neurotoxicity In primary cortical neurons, AS methanol extract greatly reduces Ab1-42 caused neurotoxicity and tau hyperphosphorylation In 2011, Zhang Z and fellow showed that AS polysaccharides (18.6% saccharose) diminish the extent of myocardial infarctions and increase cardiotrophon-1 and serum GSH levels

Immune support and hematopoiesis: AS regularly has an immunostimulatory effect, according to lymphocyte proliferation studies By initiating hematopoiesis in the bone marrow, a high molecular weight polysaccharide identified in AS has demonstrated immunostimulating activity and a blood tonifying effect This is accomplished, in part, by either direct or indirect stimulation of macrophages, fibroblasts, erythrocytes, granulocytes, and lymphocytes, and can induce an increased secretion of human growth factors from muscle tissue Hematopoiesis is further supported by the presence of significant amounts of vitamin B12, folinic acid, and biotin in AS according to the research of (Huang KC.1999)

2.2 Some kind of products in the current market

In recently, particularly in China, Angelica sinensis (Oliv.) Diels is used not only

for medicinal properties, but also as a health food product, a dietary supplement, and in cosmetics

Figure 2.3 (Dong Quai Root, 当

Radix Angelica sinensis (Oliv.)

Diels powder)

(Angelica Sinensis) Tincture, Dried

Root Liquid Extract, Dang GUI,

Glycerite Herbal Supplement 2 Oz :

Figure 2.4 Angelica sinensis

(Oliv.) Diels, Dried Root Liquid

Extract

(Angelica Sinensis) Tincture, Dried Root Liquid Extract, Dang GUI, Glycerite Herbal Supplement 2 Oz :

2.3 Fermentation technology

Fermentation is an essential metabolic process that occurs in the absence of oxygen (O2) Sugar is consumed in the absence of oxygen during the fermentation process Organic acids, gases, and alcohol are the end products of fermentation Fermentation happens frequently in yeast and bacteria, as well as in oxygen-depleted muscle cells, as in lactic acid fermentation Fermentation is a fundamental method of creating ATP by anaerobically degrading organic materials in the presence of appropriate microbes, according to microbiologist (Whitaker, A., et al.,1995)

Fermentation takes proceed in three different ways: The formation of lactic acid from pyruvate is known as homo-lactic fermentation Lactic acid, as well as other acids and alcohols, are produced during hetero-lactic fermentation The conversion of pyruvate to ethanol and carbon dioxide is known as alcoholic fermentation

2.3.1 Types of Fermentation Processes

Solid state fermentation and submerged fermentation are the two types of fermentation processes

Solid-State Fermentation: According to (Babu, et al,1996) discovered that

SSF stands for solid-state (or substrate) fermentation, which is described as fermentation that takes place in a solid, non-specific, natural condition with low moisture content Substrates such as nutrient-rich garbage can be reused in this method The solid substrates employed in solid-state fermentation are bran, bagasse, and paper pulp Because the process is slow, the substrate fermentation takes a long time As a result, the nutrients are released in a controlled manner It

is the finest fermentation technology for fungus and microorganisms since it requires less moisture This technique, however, is ineffective for bacteria since this fermentation cannot be used for an organism that requires a high level of water

Submerged fermentation: Submerged fermentation, microorganism

required a controlled atmosphere for proficient manufacture of good quality end products; attain optimum productivity and high yield Batch, fed-batch, or continuous modes are used in industrial bioreactors for the production of different types of microorganisms in a broad range (Inui, et.al.2010)

For the manufacture of alcoholic beverages (whisky, beer, brandy, rum, and wine), preservatives or acidifiers (lactic acids, citric, and vinegar) are used in food industry and for flavor enhancers (monosodium glutamate) or sweeteners (aspartate) amino acid are used in submerged batch cultivation In this part, there are different ways of submerged cultivation using microorganisms in bioreactors Here we have discussed briefly about typical features and advantages and faults of each fermentation methods are displayed Lastly, the production of microorganism

in liquid medium in various type of food industrial product has been determined

as the most important application for continuous, batch, and fed-batch cultivation (Inui, et.al.2010)

2.4 Mechanism of alcoholic fermentation

Alcoholic fermentation takes place under anaerobic circumstances in which yeast enzymes catalyze the oxidation and reduction of sugar, the main substrate in the fermentation process Before becoming active and turning the sugar to alcohol,

the yeast will need oxygen and components in the fermentation solution to develop biomass

Alcohol fermentation is a complex process that involves roughly 30 phases,

each with its own enzyme Saccharomyces (along with the majority of other

species) are involved in the fermentation process, which encompasses a series of reactions involving a variety of catalytic enzymes The main products of fermentation are ethanol, CO , and a few additional by-products A general alcoholic fermentation equation is as follows:

C H 0 + Yeast →C H OH+ 2CO +ATP (Energy)+ Heat The initial step alcoholic fermentation is the transport of sugars into the cell The three alternatives are simple diffusion, facilitated (or carrier-mediated) diffusion, and active transportation Assisted diffusion transports glucose, fructose, and mannose, which is an energy-intensive process that employs the same transportation mechanism as glucose (Jackson, 2000a) Sucrose is occasionally used for enrichment, or chaptalization, in cool-climate wine areas Because yeast cannot directly metabolize this disaccharide, invertase, an expelled enzyme, hydrolyzes it outside the cell The monosaccharides that arise (glucose and fructose) are then transported into the cell The resulting monosaccharides (glucose and fructose) are subsequently carried into the cell (Jackson, 2000c) After glucose

is transformed into cell yeast the glycolysis should be embraced:

Glycolysis is the most prevalent mechanism for glucose (as well as fructose and mannose) breakdown, and its primary function under anaerobic environments

is energy generation When organisms capable of fermentation, such as S cerevisiae, become anaerobic, the rate of glycolysis increases by 10 times or more, allowing for a growth-sustaining level of energy generation despite the poor yield

of ATP created by glucose metabolism through this route (Purves et al., 2001) Parts of the initial glucose carbon skeleton will be utilized for biomass formation

or anabolism during glycolysis (the synthesis of biological molecules, amino acids, nucleic acids, lipids etc., according to the need of the growing and multiplying cells) Glycolysis is made up of ten stages, each of which is catalyzed by a different enzyme

In yeast cells, glucose interacts with the phosphate moiety of ATP (adenosintriphosphat) to create gluco-6-phosphate and ADP, which is mediated by the Hexokinase enzyme (Kuser et al., 2008)

C H O + ATP →CH O H P0 CHOH CHO + ADP

After that glucose-6-phosphate by the action of phosphoglucoisomerase enzyme will turn into fructose-6- phosphate

CH O H PO CHOH CHO→CH O H PO CHOH COCH OH

The second ATP molecule will connect another phosphate moiety to fructose

- 6 - phosphate under the action of phosphofructokinase, forming fructose diphosphate and the second ADP molecule:

1–6-CH O H PO 1–6-CHOH CO1–6-CH OH + ATP

→ CH O H PO COCH O H PO + ADP The enzyme aldolase then splits the six-carbon sugar into two three-carbon moieties (glyceraldehyde and dihydroxyacetone), completing the preliminary processes (Jackson, 2000b)

CH O H PO CO CHOH CH O H PO

→ CH O H PO COCH OH+ CH O H PO CHOHCHO

Aldehydes and glyceric phosphates have a major part in the later transformations of alcoholic fermentation, while they play a minor function in the fermentation solution Isomerism, which is caused by the enzyme triphosphate -isomerase

CH O H PO COCH OH →CH O H PO CHOHCHO Following that, dihydroxyacetone is transformed to glyceraldehydes, and the two glyceraldehydes molecules are oxidized (hydrogen atoms are stripped of their electrons), reducing NAD to NADH (Jackson, 2000b)

2CH O H PO CHOHCHO+2H PO + NADH

→ 2CH O H PO CHOHCOO+H PO +2 NADH +2H

According to (Jackson, 2000b) revealed that the high-energy phosphate moiety will be transferred to the ADP molecule in the next stages, with the aid of phosphoglyceratekinase 3-phosphoglyceric is generated as a consequence, while ADP obtains more energy and transforms it to ATP The phosphoric acid radical

will shift from the third to the second carbon position due to the action of the enzyme phosphoglyceromutase As a result of the process, 3-phosphoglyceric converts into 2 phosphoglyceric:

2CH O H PO CHOHCOOH→2CH OHCHO H PO COOH

Enolase enzyme causes 2-phosphoglyceric acid to lose water and convert to phosphoenolpyruvic acid:

2CH OHCHO H PO COOH→ 2CH OH H PO COOH+ 2H O

Because phosphoric acid is so unstable, the phosphoric acid pyruvate kinase radical is easily lost, resulting in the formation of pyruvic acid

2CH = CO− H PO COOH + 2ADP → 2CH CO − COOH + 2ATP Pyruvic acid is transformed to acetaldehyde in this phase, which produces carbon dioxide as a byproduct

2CH CO − COOH → 2CO + 2CH CHO The final stage of alcohol fermentation is that acetaldehyde is reduced by NADH

life (Shiby and Mishra, 2013)

In these day, males consume around 5000 different types of fermented foods all over the world The majority of them are ethnic in nature and produced in small quantities to meet the demands of distinct populations in a given place Fermented food preparation has expanded over time to include a variety of locally available substrates such as vegetables, grains, seafood, dairy, and meat Fermented meals have a better flavor, texture, and color, which is why rural people prefer them to unfermented foods (Sekar and Mariappan, 2007)

Beverages are an important part of the food industry for human consumption Ordinarily, beverages are divided into two categories: alcoholic and non-alcoholic Soft drinks, such as juice, tea, and coffee, as well as carbonated and non-carbonated drinks, are nonalcoholic beverages Beer, wine, and spirits are examples of alcoholic beverages The dairy industry manufactures a variety of liquid dairy products, including milk, a natural beverage All fermented dairy products are derived from the milk of practically all domesticated milking animals; nevertheless, a number of innovative fermented milk product variants have been developed, and they are gaining popularity around the world

Apple cider, mead, rice sake, grain beer, and grape wine (the most popular beverage) are all examples of fermented liquids that include sugar (Hornsey, 2007) Wild berries, various fruits, sap, honey, and animal milk come to mind when thinking about the primary sources of sugar available in the past The activity

of natural flora already present in the meal or those introduced from the environment causes traditional fermentation Scientists have also succeeded in identifying and defining the bacteria that execute this type of fermentation

2.5.2 Historical development of fermented beverage

Fermented beverages have been consumed for several thousand years all over the world, and for most of that time the fermenting microorganisms were not known at all The traditional fermentation processes occurred by spontaneous, mixed culture fermentation of carbohydrate-rich substrates The achieved fermentation products are strongly dependent on the substrate, the naturally occurring microorganisms involved in the fermentation and the fermentation conditions, such as temperature, pH, and oxygen availability Fermented beverages exhibit several beneficial properties like attractive flavor, good digestibility, increased nutritional value, and a reduction of pathogenic microorganisms (Bourdichon et al., 2012) The latter led to the utilization of beer as a secure beverage, as compared to water in medieval times The explanation for this started

to develop with the discovery of microorganisms by Antonie van Leeuwenhoek in

1683 Afterwards, it took nearly 200 years until Louis Pasteur discovered in 1857 that yeast is responsible for alcoholic fermentations This knowledge was applied

by Max Emil Julius Delbrück, who developed the first pure yeast culture in 1895

The sterile fermentation of pure cultures offers the possibility of developing controlled processes as they are applied in modern beer brewing processes While pure culture processes lead to reproducible fermentation products with constant flavor quality, predictable shelf life, and consistent texture or “mouthfeel,” especially in beverage production, there still exist many fermentation processes that rely on spontaneous mixed cultures or cocultures, such as wine or Belgian beers

well-The amount of different microorganisms in mixed fermentations offers the advantage of producing many different flavor-active substances, resulting in a huge variety of flavor profiles Especially in wine production, different flavor profiles are favored and the quality of fermentation products alters between location and year These differences can be minimized when the fermentation is inoculated with a high concentration of a single microorganism for example,

flavor complexity A compromise between a controllable process and a rich flavor profile can be the application of the controlled coculture process In this case, inoculation is conducted with a mixture of selected microorganisms (yeasts and bacteria) Undesired microorganisms are not able to proliferate and simultaneously many flavor active substances are produced Furthermore, alcohol content of the fermentation product can be influenced by the applied microorganisms

2.5.3 Classification of fermented beverages

Fermented beverages can differ based on the type of food prepared, the fermentation process' time, and the addition of microorganisms Fermented beverage encompasses alcoholic beverage and dairy beverages (Kumar Anal, 2019)

Alcoholic beverages including wines, beer, cider, spirits, mead, sake and other On the other hand yogurt, cultured buttermilk, and sour cream, Kefir, koumiss, Yakult, and others are example of dairy beverage

2.5.4 Health benefits of fermented beverages

Fermented beverages have a number of advantages in addition to their nutritional value In general, fermented foods are easier to digest than unfermented foods As a result, fermented foods are employed as weaning foods in a variety of cultures Acid is created slowly, allowing for the formation of tiny particles during fermentation It also aids in the expansion of the surface area available for later human enzyme digestion Traditional goods have been considered as a treatment for intestinal, stomach, and liver, as well as for boosting the appetite, for decades before the discovery of bacteria and other microorganisms In fermented beverage the numerous health effects for probiotics have been established, which include alleviation of the symptoms of lactose intolerance, decrease of a period of rotavirus diarrhea, diclined in detrimental intestinal microbial enzyme activities, diminished fecal mutagenicity, and immune improvement

According to Marquina et al (2002), kefir consumption significantly increased LAB counts in the intestinal mucosa and reduced Enterobacteria and Clostridia populations It also prevented Campylobacter jejuni colonization in chick ceca (Zacconi et al., 2003) It was also found effective in patients with gastrointestinal disorders and in postoperative treatments (Sarkar, 2007) Researchers have used kefir in Russia in the treatment of peptic ulcers in the

stomach and duodenum of human patients (Farnworth, 2008)

Probiotics have been shown to have a variety of health benefits in fermented beverages For example: Including relief of lactose intolerance symptoms, reduction in the duration of rotavirus diarrhea, decrease in harmful intestinal microbial enzyme activities, decreased fecal mutagenicity, and immune improvement Another example is Kefir consumption increased LAB counts in the intestinal mucosa and diminished Enterobacteria and Clostridia populations,

according to Marquina et al (2002) Campylobacter jejuni colonization in chick

ceca was also inhibited (Zacconi et al., 2003) It has also been reported to be beneficial in the treatment of gastrointestinal diseases and postoperative care (Sarkar, 2007) Kefir has been utilized in the treatment of peptic ulcers in the stomach and duodenum of human patients in Russia (Farnworth, 2008) Another example is red wine drinking may provide an additional preventive impact against

cardiovascular diseases (CVDs) when compared to other alcoholic beverages In a research, 10 healthy individuals were given dealcoholized red wine, or gin for 20 days, and the results revealed that red wine polyphenols can inhibit non-beneficial bacteria in the human microbiota and potentiate the growth of probiotic bacteria like Bifidobacteria, which could be linked to lower C-reactive protein (CRP) and cholesterol levels

2.6 Low alcohol beverage

Low alcohol is defined as a fermented beverage having a maximum alcohol

by volume (abv) of 1.2 % or less, and the maximum abv should be stated on the

label (Guide on Labelling Low and No Alcohol Products Designed as “spirit

Because of the growing awareness of well-being and health, consumers are becoming more interested in low-alcohol drinks (BLA) or nonalcoholic beverages (NAB) Kombucha, Hardaliye (a combination of grapes, mustard seeds, and sour cherry leaf), Sima (fermented lemonades), and Kvass (a combination of rye or barley

in the form of malt or baked bread), as well as alcohol-free wine, are examples Low alcohol has good preservation capabilities ("shelf life") and, in many situations, the drinking of these fermented beverages has health benefits For example, decreased risk of cardiovascular disease, may help boost metabolism, aid depression treatment, lower blood sugar, and maintain a healthy weigh Aside from the aforementioned alcohol-free beverages, there is a growing desire for alcohol-free beverages with sensory qualities equal to those of alcoholic beverages This demand is fueled in part by many customers' growing health concern, as evidenced

by the increased demand for wine, beer, and sparkling wine As of now, the adoption of alcohol-free versions of these beverages is minimal, owing to the fact that they often do not taste as well as the alcohol-containing version The low alcohol variant is often sweeter and has less aromatic fermentation products Many alcohol-free beers have a delectable off-flavor (Missbach et al., 2017) For the manufacturing of low-alcohol beverages and non-alcoholic beverages, many biological and physical processes are used Fermentation time reduction is one biological approach for reducing the amount of ethanol produced The microbial conversion of the substrate is terminated quickly after inoculation with

the yeast in this stalled or limited fermentation process As a result, there is a strong flavor and a lack of flavor ingredients that are desired (e.g., aldehydes and aromatic compounds) Furthermore, the product has a high concentration of fermentable sugars and a pH value that is comparable to that of wine Both of these events could lead to a decline in the finished beverage's microbiological stability.

2.6.1 The method used in the low-alcohol production

Several approaches can be used to create low alcohol levels Evaporation, distillation, dialysis, reverse osmosis (RO), and pervaporation (PV) were among the methods used

The first method is evaporation: Ethanol may be removed from beverages

by evaporation because of its low boiling point However, there are a number of drawbacks to this method Chemical reactions occur as a result of the high temperatures, resulting in a change in the beverage's flavor In addition, the beverage's aroma-active volatile components are eliminated Evaporation, on the other hand, necessitates a lot of energy Because of these drawbacks, this approach

is not utilized to reduce the amount of alcohol in beverages The heat effect is reduced when vacuum evaporation (VE) is used On an industrial scale, this process is utilized to produce alcohol-free beer (Brányik et al., 2012)

The second method is a distillation: This technique, as opposed to evaporation,

can eliminate the ethanol fraction Compounds that are desired (for example, flavor and fragrance compounds) can be directly added to the beverage Vacuum distillation (VD) at temperatures between 30°C and 60°C can minimize the needed temperature (Brányik et al., 2012) This reduces the amount of energy required for the procedure while also preventing undesired alterations in taste components The total removal of ethanol from the beverage, the ability to sell the extracted ethanol, and the ability to automate the process are all advantages of this method (Brányik et al., 2012) The spinning cone column (SCC) is one of the most widely used processes for producing alcohol-free or low-alcohol wines today

The next method is membrane-based methods this method encompass the

dialysis/diafiltration and the RO The dialysis/diafiltration and RO procedures are included in the membrane-based methods method They have the benefit of having

a low energy demand Furthermore, there are no thermally induced chemical

reactions that are undesirable As a result, when ethanol is eliminated using membrane-based technologies, taste stability is good The membrane that has been applied serves as a molecular sieve The membrane allows water and ethanol to travel through it Filtration is done under pressure to avoid water osmosis from the dialysate into the beer The dialysate is permeated with ethanol Effective ethanol removal can be achieved if the procedure is run in counter-current modes (Brányik

et al., 2012)

The following method is pervaporation this method concludes: permeation

and evaporation This method has great promise for the separation of challenging mixtures because of its excellent selectivity (Roy and Singha, 2017) Since the product does not require heating, there is no risk of flavor characteristics being altered as a result of chemical reactions Pervaporation is an excellent technique for reducing chemicals that are present in low concentrations As a result, it can be used in the food industry to recover fragrance compounds The method is very reliant on a good membrane, and there are numerous publications that go through various pervaporation materials in depth Permeation through the membrane can take place in either a passive or active manner Concentration gradients between the feed and the permeate drive passive permeation A chemical interaction occurs between the penetrating substance and a membrane component, resulting in active transport (Roy and Singha, 2017)

Pervaporation membranes had hitherto been prohibitively expensive This prevents them from being used on a large scale, but there have been considerable advancements in the technology, such as better membrane stability Pervaporation

is projected to be used in many industrial processes in the next years due to its great selectivity and the fact that it requires no thermal input (Catarino and Mendes, 2011) propose a two-step procedure for de-alcoholizing beer Pervaporation is used in the initial stage to extract volatile chemicals, which are then collected The spinning cone column is used to eliminate ethanol from the retentate in the second stage The scent compounds that were caught in the first phase are then reintroduced into the retentate In addition, some untreated beer is combined with alcohol-free beer to produce a beer with a low alcohol concentration of 0.5% (v/v) and a balanced flavor profile

2.7 Saccharomyces bayanus

subsp uvarum) is a yeast belonging to the genus of Saccharomyces that is

employed in winemaking, cider fermentation, and the production of distilled

drinks Saccharomyces bayanus, like Saccharomyces pastorianus, is recently taken to the result of several hybridization events between Saccharomyces uvarum,

investigations, and most commercial yeast cultures labeled as pure S bayanus for

winemaking generated a comprehensive resource for expression profiles for

method, which has been used in various comparative investigations in yeast systems, including expression patterns and nucleosome profiles (Caudy et al.,

2013) Unlike S.cerevisiae, S Bayanus is typically galactose negative

2.7.1 Morphology

unicellular organism that is ellipsoid or ovoid in shape and ranges in size from 5

to 14 micrometers Saccharomyces bayanus reproduces through budding in the cell

according to (Oca et al., 2016)

2.7.2 Structural

phylum Ascomycota, and kingdom Fungi The following are the key components

contains information and is found in the cytoplasm In the process of cell biosynthesis and reproduction, heterologous messengers for cells and related components are used (Oca et al., 2016)

2.7.3 Chemical components

on the culture medium's conditions, the nutrients in the medium, and the physiological state of the cells 70-75 % of pressed yeast is water, with the remaining 25-30 % being dry matter Water refers to both outside and inside the cell Depending on the yeast strain, culture methodology, and cell collecting method, the amount of water used varies For instance, when cells are grown on NaCl media, the amount of water in the cell drops (Yamada & Sgarbieri, 2005)

The inorganic matter: Proteins and other nitrogenous compounds constitute for 50% of yeast cell composition, followed by fat (1.6%), carbohydrates (33.2%), cell tissue (7.6%), and ash (7.6%) These compounds' composition is not permanent; it can alter during culture and fermentation

Carbohydrates contents: Polysaccharides, glycogen, trehalose (12-12.5%), mannan (18.7-24.9%), glucan (9.47-10.96%), and chitin

Glucan, mannan, and the alkaline and hydrochloric acid forms of glycogen are structural constituents of the cell, while trehalose and glycogen are soluble, according to kinetic studies of carbohydrate transformation during yeast storage The main source of energy for cells is acid acetic The amount of trehalose in yeast

is related to its stability as the more trehalose there is, the more stable the yeast (Yamada & Sgarbieri, 2005)

Glycerol, phosphorus lipid, free sterols, and a variety of sterols and esters make up yeast fat In yeast, the quantity of ash comprised 6.5-12% of the dry matter and varies depending on the strain

2.8 Microbiology in fermented beverages

Yeasts, molds, bacteria, enzymes, and other microorganisms are among the microorganisms involved in the fermentation process This microbiome is present

in all of the materials, including production tools, the outside environment, atmosphere, and etc (Pretorius, 2000)

2.8.1 Yeast

Yeasts are eukaryotic microorganisms that are important components in the fermentation process belonging to the kingdom Fungi Myriad studies discovered

that some yeast, such as Saccharomyces cerevisiae, Saccharomyces bayanus, or

based on the structure and ability to ferment, generated alcohol concentration, and

specialized odor Saccharomyces cerevisiae has been employed for thousands of

years in both baking and alcoholic beverage manufacture There are several strains

of Saccharomyces cerevisiae, with variances in the known yeasts: wine yeast,

brewer's yeast, mesta yeast, growth yeast, bottom yeast, yeast juice, and yeast is the dominant, and sometimes the only, species that survive in wine fermentation, with most others dying after a few days due to its combination of effective fermentative catabolism and relatively strong alcohol tolerance (Pretorius, 2000)

According to (Gancedo et,al 1989) Saccharomyces cerevisiae is a facultative

anaerobic yeast that reproduces through budding and generates invertase, an enzyme that can break down sucrose into fructose and glucose Numerous studies

demonstrate that Saccharomyces cerevisiae can ferment juices with high sugar

content and create 18 to 20% (v/v) alcohol content, thrives at pH 3.75 to 4.5, has the ability to precipitate, providing flavor, and has a very nice characteristic taste

for Saccharomyces cerevisiae in wine As a result, Saccharomyces cerevisiae is

frequently used to ferment wines with a high alcohol level

Yeast requirement in fermented beverages

• The yeast for fermentation must have high fermentation ability, rapid growth rate, budding rate of 10-15%, and dead cell count of less than 2% and being able to ferment at a reasonably high temperature (> 35 C), with an alcohol concentration ranging from 7 to 16% after fermentation

• The yeast for fermentation could ferment a variety of sugars, resistant to sugar concentrations, alcohol, environmental acidity, and high antiseptics (fermented in an accessible medium of 8 to 12% alcohol) (Vandamme & Derycke, 1983) has good settling ability, resulting in a product with increased sensory value

2.8.2 Lactic acid bacteria

Lactic acid bacteria are present in all fermentations to variable degrees, influencing the final result in a good or negative way It's caused by unfavorable yeast, for instance soybean isn't a good substrate for lactic acid bacteria to grow

on Lactic acid bacteria frequently deteriorate the quality of alcoholic fermentation

products The alcoholic fermentation of cereals, on the other hand, transmits Lactic acid bacteria in the early stages of fermentation generate a suitable environment for subsequent fermentation, including alcohol production, contributing to the distinctive flavor and aroma of drinks Lactic acid bacteria play an important part

in the fermentation of vegetable-derived raw materials, and the optimal amount of acid generation varies depending on the product type Lactic acid bacteria, on flip side, are significant for two reasons: they influence wine quality and cost of production according to (Wibowo et al., 1985)

Lactic acid bacteria are Gram-positive, catalase-deficient, and fastidious They need nutrient-rich media and flourish on glucose-yeast-extract agar, but are stymied by penicillin and they are smaller than yeast In terms of structure, lactic acid bacteria are unicellular organisms that are fermentable, resistant to gases, high acidity, and sulfur dioxide in solution Malolactic bacteria are types of bacteria that prefer to metabolize malic acid rather than carbohydrates or citric acid and are more resistant to lower pH levels Sugar usage by lactic acid bacteria in wines has revealed that while there a species and strain distinctions in sugar utilization, however, various species can metabolize pentose, tartaric acid, and glycerol

(Wibowo et al., 1985)

They are also divided into two groups homofermentative and heterofermentative For homofermentative bacteria produce 80-90% lactic acid from glucose, while heterofermentative bacteria produce 30-50% lactic acid from glucose and also convert citric acid to acetic acid Homofermentative cocci encompass Lactobacillus plantarum and Lactobacillus casei, while

heterofermentative cocci include Lactobacillus buchneri, Lactobacillus brevis,

molactic fermentation

Aside from the fact that Leuconostoc oenos has been reclassified as

over other lactic acid bacteria due to its acid tolerance and flavor profile It thrives

in low-oxygen environments Oenococcus oeni has adapted to high ethanol

concentrations (<15% (v/v)), low pH (as low as 2.9), and limited food availability

unreliable organism to work with It's a picky creature that requires group B

vitamins, organic nitrogenous base, and 11 amino acids, the majority of which can

be obtained through yeast autolysis Growth will be stifled if any of these nutrients are missing (Wibowo et al., 1985)

Table 2.1 Several microbes play a significant role in fermented beverage

(Yamada & Sgarbieri, 2005)

Saccharomyces cerevisiae var,

additional color and flavor

Acetic bacteria and

2.9 Some factors affect to fermented beverage

2.9.1 Influence of temperature

The temperature of fermentation has a considerable effect on the yeast's life, the fermentation process, and the final product's quality Myriad of research on wine production have found that the best temperature range for wine yeast is between 25-32°C Yeast is less active and weaker at lower temperatures However, certain strains of low-temperature resistant yeast can ferment at 4-10°C, but only slowly and for a long time, resulting in a huge quantity of residual sugar in the fermentation medium, which is an ideal habitat for pathogenic microbes to thrive The temperature may be raised to 36°C and the yeast will still function correctly, but it will not work above that degree (Torija et al 2003b)

At 38°C, the enzyme begins to be inhibited; at 40°C, it practically becomes inactive, and at 40°C, it progressively dies For instance on the aromatic quality of the wine Low temperatures and a slower fermentation are known to help preserve the juice's rich tastes These scent compounds are destroyed at high temperatures White wine, for example, (e.g Reisling, Gewürztraminer, Traminette, Vidal Blanc) should be fermented at cool temperatures to avoid volatile fragrance components from 'boiling' when carbon dioxide is released quickly The quantity

of ethanol and glycerol produced is likewise dramatically reduced at higher temperatures (Beltran et al., 2002b)

On the other hand, Red wines, should be fermented at higher temperatures to enhance tannin extraction from skins The lowest required temperature for red wine is 20°C, with ideal temperatures of 25–30°C and maximum temperatures of 32°C (Molina et al., 2007)

The effect of fermentation temperature on the generation of yeast-derived aroma molecules at 15°C and 28°C was examined in a study by (Molina et al.,2007) Fresh and fruity scent components were identified in higher quantities at 15°C, whereas floral related aroma compounds were detected in larger concentrations at 28°C The rate of synthesis of volatile scent compounds differed depending on the development stage Additionally, linear connections between increases in higher alcohol concentrations and their equivalent acetates were discovered These findings show that fermentation temperature has a significant

impact on the wine's ultimate fragrance profile and is thus an essential control parameter for fine-tuning wine quality during the winemaking process

Temperature has an impact on the extraction of phenolics and color in red wine fermentation When the rate of fermentation is high, yeast growth and metabolism are boosted, which raises the temperature even more, resulting in a quicker rate of fermentation If the heat generated by this process is allowed to continue, the yeast may be killed Large-scale fermentations with insufficient cooling at high ambient temperatures are more prone to this issue It can happen

in smaller fermentations if the cooling system fails Early detection allows for early action, improving the odds of effectively boosting the fermentation rate and preventing the fermentation from stopping Fermentations that are overly sluggish,

on the other hand, are inconvenient and increase the likelihood of considerable acetic acid production (Molina et al (2007)

2.9.2 Influence of oxygen on fermentation

Oxygen is essential not only for yeast reproduction and optimal development, but also for fermentation efficiency and product quality In term of yeast reproduction In fact yeasts require oxygen to manufacture and ingest essential compounds for reproduction, and if the must is not properly aerated at the onset of fermentation, oxygen will become the limiting factor for yeast quantity (Mauricio

et al., 1998)

Cell metabolism can be changed in the lack of oxygen at the outset of the fermentation process to promote the formation of certain aroma compounds, notably esters and higher alcohols (Pretorius and Lambrechts, 2000) The presence

of oxygen during the early stages of fermentation appears to boost yeast survival throughout the later stages of fermentation, resulting in less residual sugar in the final wine This might be explained by the partial elimination of harmful medium-chain (C8–C12) fatty acid chains and the increased synthesis of long-chain (C16–C18) fatty acids and sterols, both of which contribute to improved sugar absorption across the cell membrane

As a result, the proper amount of oxygen for fermentation will help satisfy the early needs for yeast reproduction and vigorous growth However, if there is

too much oxygen in the air, the fermentation process will be slowed down and the fermentation efficiency will be reduced

2.9.3 Influence of pH

The most essential element affecting product formation is pH, which has a significant impact on the fermentation process It influences yeast activity because it allows them to alter the size of the cell membrane, enhance or decrease nutrient permeability, and change the direction of fermentation (Narendranath & Power, 2005)

Yeast can grow in a pH range from 2 to 8, however the pH range between 4

- 4.5 is the most favorable and powerful for growth When the pH is too low or too high, it interferes with the fermentation process, allowing dangerous microbes to thrive It is extremely simple to become contaminated, especially if the pH is raised; glycerol will produce more and the fermentation efficiency will be reduced (Narendranath & Power, 2005)

As a result, any acid can be added to the fermentation medium to establish appropriate pH conditions as long as the anion of the acid does not have a severe effect on yeast activity

2.9.4 Influence of sugar concentration

Sugars are significant because yeast cells ferment them and convert them to alcohol and carbon dioxide Sugars also give wines with residual or added sugar a sweet flavor Sugar concentrations are measured as must density, and expressed as different units in different wine producing regions (Learmonth, 2016)

A sugar concentration that is too high will cause the pressure to rise, resulting

in a loss of equilibrium, the physiological state of the yeast, and the quality of the product Additionally, glucose metabolism yields largely ethanol, and oxidative sugar metabolism is unlikely The O mediated suppression of glycolysis (the so-called Pasteur effect) occurs only when the substrate concentration is low (less than 2 g/L)

Sugar can either speed up or slow down the fermentation process Reduced sugar concentration, on the other hand, causes low ethanol production It was discovered that the optimal concentration of fermentation medium for wine yeast

to function is between 10% and 25% (Learmonth, 2016)

Fermentation is slow and difficult if the concentration is greater than 25% If the fermentation medium concentration is greater than 35%, it will virtually completely stop the fermentation process The amount of sugar in the fermentation medium has a significant impact on the rate and efficiency of fermentation In different wine-producing regions, sugar concentrations are measured as must density and represented in different units

Sugar concentrations are frequently measured in Europe in Oeschle (related to the specific gravity of must; actually, ◦Oe = [specific gravity – 1.00] 1000) and in the New World in ◦Brix or ◦Balling (both a measure of percent w:v sugar) Sugar concentration in physiologically mature grapes can range from 18 to 26 ◦B (g/100 g), but sugar quality in over-ripe and botrytized grapes

can be well over 30◦B (Spedding, 2016)

2.9.5 Influence of yeast ratio on fermentation medium

The fermentation process is carried out by yeast, which converts carbohydrates into ethanol and CO The amount of yeast supplied to the fermentation medium has a significant impact on the process The fermentation process runs smoothly, the recovery efficiency is great, and the product quality is also higher when the ratio of yeast used is appropriate

When the amount of yeast introduced is modest, the fermentation process is delayed, which allows microorganisms to multiply more easily If there is too much yeast added, the fermentation medium will be insufficient for the yeast to work and proliferate as a result yeast will slowly die, and the product will have a smell peculiar, non-specific odor In general, 6-10 million cells/ml of fermentation medium is an appropriate amount of yeast to add to the cell fluid (Learmonth, 2016)

2.9.6 Influence of alcohol content

There are yeast variants that tolerate alcohol at low concentrations, such as

yeast strains, such as Saccharomyces cerevisiae, can withstand 9%-12 % (v/v) alcohol

and ferment up to 14-16 percent Increasing the concentration will impede not only the live activities of yeast, but also the activity of several enzymes that convert sugar

to alcohol during alcoholic fermentation (Tian et al., 2021)

2.9.7 Influence of light

Light is one of the factors that affects the fermentation process and can limit yeast cell activity The UV radiation, in particular, will harm yeast cells As a result, it must be fermented in the proper light weather (Shu et al., 2009)

2.9.8 Influence of organic acids

Several studies have shown that acid is a crucial component in lowering pH and preventing the growth of a variety of bacteria Organic acids influence fermentation not because of the total sugar content of non-volatile organic acids, but because of the composition and content of some organic acids, fatty acids, acid acetic, butyric, propionic, present in the fermentation medium, which will influence yeast reproduction and growth (Chidi et al., 2018)

2.9.9 Influence of minerals

According to (Obiajunwa et al., 2002) have shown that elements like N, P, S, and others are vital in the creation of the cell membrane and nucleus, and that Cu,

Ca, and Mg are the activation centers of many enzymes involved in the conversion

of sugar to alcohol Cu is plentiful in polyphenol oxidase, and Mg is abundant in enzyme cofactors Furthermore, salts such as P, K, and others operate as a buffer solution to maintain the pH of the environment Mineral salts required for alcohol fermentation are often found in the following sources: Mg, K, I2, Ca, Co, Fe, S,P, In addition, some vitamins, such as B1, biotin, vitamin E, and so on Also has

a very positive effect on yeast reproduction and growth

2.10 By-products of fermentation

In the alcoholic fermentation process, in addition to the two main products, ethanol and CO , there are also by-products such as organic acids (acetic acid, lactic acid, citric acid, pyruvic acid), glycerol, acetaldehyde, aceton, diacetyl, and ester (Ferreira & Mendes-Faia, 2019)

2.10.1 Formation of organic acids

Acetic acid is created when two acetic aldehydes undergo a redox reaction One molecule is oxidized, while the other is reduced

CH CHO + CH CHO + H O−> CH COOH + C H OH Lactic Acid: Pyruvate dehydronase produces lactic acid in the winemaking process by the following reaction (Ferreira & Mendes-Faia, 2019)

CH CHOCOOH + NAD H → CH CHOHCOOH + NAD

2.10.2 Glycerol

Glycerol is produced not just by yeasts, but it can also be used as a carbon source in aerobic cultures The primary function of glycerol synthesis during wine fermentation is to provide an osmotic stress-responsive solution to the yeast cell and to equilibrate the intracellular redox balance by converting excess NADH

created during biomass production to NAD+ (Pretorius, 2000)

The precursor of glycerol, glycerol-3-phosphate, is an important step in the formation of membrane lipids Glycerol is viscous and has a little sweet taste and

it gives the product smoothness, consistency, and general body, therefore it is usually considered as a positive contribution if it is created abundantly during fermentation The amount of glycerol produced by yeast during fermentation depends on the nitrogen content, sugar content, sulfur content, and pH of the grape must, as well as fermentation temperature, aeration, starter yeast strain, and inoculation level Furthermore, pH has an impact on the glycerol synthesis process It was discovered that the higher the pH, the more sugar was involved

in the synthesis of glycerol Glycerol concentrations in red wines are often higher than in white wines under controlled conditions, ranging from 1 to 15 g/l Typically, about 4–10% of the carbon source is converted to glycerol, resulting in glycerol levels that are 7–10% that of ethanol

A growing demand for table wine with lower ethanol levels could be met by yeast overproduction of glycerol at the expense of ethanol Wine yeasts with a reduced glycerol pathway, on the other hand, would produce more alcohol, which would be beneficial for the production of brandy and other distilled products

(Pretorius, 2000)

CHAPTER III MATERIALS, RESEARCH CONTENTS AND METHODOLOGY 3.1 Material and research scope

3.1.1 Material

two years of cultivation This root will be cleaned and removed from broken slices After that this material have been drying for 8 hours at 60°C until the moisture content reaches under 10%, and macerated with 70% (v/v) ethanol for 12 hours at

45°C to get the total polysaccharides The residue of Angelica sinensis (Oliv.)

Diels root was dried and kept under 13% after that preserved in PE plastic bags

and used for this research

and food technology at Hanoi University of Science and Technology

3.1.2 Research scope

Research was carried out in the laboratory scale

3.2 Work place and time to proceed

- Location: Laboratory of Department of Food Technology, Faculty of Biotechnology and Food Technology, at Thai Nguyen University of Agriculture and Forestry

- Implementation time: February 2021 to February 2022

3.3 Chemicals, equipment

Table 3.1 Experiment chemicals

Table 3.2 Laboratory instruments

No Experimental equipment Origin of products

digestion and distillation set,

Germany