Wine is the world's largest production and most popular single-sugar brew. It is rich in nutrients and has obvious health effects. However, the fermentation process of wine is complex and difficult to control, and it is easy to produce potentially harmful substances biogenic amines. This is a class of nitrogen-containing, small-molecule alkaline organic compounds produced by organic organisms that are often detected in alcoholic beverages. Excessive amounts of biogenic amines can cause a variety of adverse reactions in the human body, and in severe cases, can be life-threatening. Therefore, degradation of biogenic amines in wine is necessary. We designed a Saccharomyces cerevisiae strain with high production of amine oxidase to cleave biogenic amines to effectively control the risk of biogenic amines in wine. We first studied the basic information and the overall market. Then, we conducted expert interviews and field research. Finally, we conducted different online and offline socio-educational activities with the aim of informing people about biogenic amines and the functions of our product.

Figure1：Mind map of IHP

We have received a total of 206 valid responses to our questionnaire. Here is the analysis of the questionnaire results:

Figure 2. Question: What is your gender?

Figure 3. Question: Your approximate income level is?

Figure 4. Question: What is your age?

To ensure that our research is objective, the distribution of respondents to the survey needs to be even. So we started by surveying people's gender, age group, and income level. The results of the study showed that the ratio of men to women in our study was 48.54%:51.46%, a relatively even figure. Most of the people's income levels are concentrated in the middle income (4500-15000 yuan per month). In terms of the age distribution of the study population, about 50% of the people are over 35 years old and about 50% are under 35 years old.

Figure 5. Question: Of the various types of alcohol listed below, which do you drink most often?

To find out the popularity of wine among the masses we did a survey. The results showed that 48% of people chose beer, 28% chose wine, 22% chose liquor and 1% chose yellow wine. According to the above data, we can understand that red wine is still very popular among consumers in the alcohol sales market. Since our products are mainly aimed at red wine, our market should also be positively affected.

Figure 6.

Question: Do you think wineries should indicate the number of biogenic amines in their wines on the label?

In order to investigate the importance of health for consumers in the market, we designed questions accordingly. The results of the survey showed that 96.12% of the people thought that wine producers should label the biogenic amine content in wine on the bottle before leaving the factory. This result shows that once consumers are aware of the harmful effects of biogenic amines in wine, most of them would like to know the biogenic amine content before buying wine to ensure their health. This shows that our product has a very promising market potential.

Figure 7. Cross-tabulation of income status and alcohol consumption

Figure 8. Cross-tabulation of income status and frequency of alcohol consumption

In order to understand issues related to the consuming population, we cross-tabulated people's income with the amount and frequency of alcohol consumption. The results show that among consumers of all income levels who drink red wine more frequently and in larger quantities are dominated by people with higher incomes. Through this survey, we were able to see that our products may be targeted to the higher income groups in the future.

Figure 9. Cross-tabulation of people's income levels and how much they are willing to pay a premium for healthier products.

In order to determine what consumers expect from our new product, we asked them how much of a premium they would be willing to pay in their minds to purchase our product. The results show that every income level is willing to buy our product if the premium range is kept below 40%. Therefore, we can achieve relatively good sales results by keeping the premium of our product within 40%.

Figure 10. Question: Do you want to reduce the biogenic amine content of alcohol to minimize the negative effects of alcohol on your body?

We designed this question in order to understand consumer attitudes toward our products. The results showed that a whopping 93.69% of people would like to use our products to protect their health in the future. So we can conclude that our product has a long-term future and is also very influential.

3.1 Expert Introduction

In order to learn more about biogenic amines and healthy drinking, we are honored to have Prof. Li Qi to advise us on our project topic, specifically focusing on the "amine" Saccharomyces cerevisiae. Prof. Li Qi is from Sichuan Normal University and graduated from the Center of Excellence in Molecular Plant Science of the Chinese Academy of Sciences with a PhD degree in 2019. Her research interests includes microbial genome editing, microbial resource mining for Sichuan wine brewing, and microbial synthetic biology.

Figure11: we are interviewing Dr. Li

3.2 Interview overview

(1) Factors affecting biogenic amines

During the interviews we learned that it is extremely important to control the level of biogenic amines during the manufacturing process of fermented foods. It is common for wines to contain about 60 to 70 mg of biogenic amines per liter. Biogenic amines are similar to vitamins in that exceeding a certain amount may have adverse effects on the human body. In specific biogenic amines, such as histamine, consuming more than 100 mg may result in symptoms of toxicity. Degradation of biogenic amines has little or no effect on the taste and flavor of wine.

The three main factors affecting the production of biogenic amines are, microorganisms, raw materials and fermentation conditions. In order to reduce the biogenic amine content in food, low-temperature storage, floatation techniques and processing techniques can be used to help slow down the growth of microorganisms or degrade biogenic amines. Temperature regulation can optimize degradation. Degradation of biogenic amines can also be facilitated by modified fermentation strains, especially Saccharomyces cerevisiae strains carrying amine oxidase, but the safety of the strains needs to be evaluated. In daily life, easy detection methods for biogenic amines include color development methods or kits, which help the average consumer to know the amount of biogenic amines in food.

In summary, the formation of biogenic amines involves several factors, and a comprehensive strategy can effectively reduce the biogenic amine content, thus improving the safety and quality of food.

(2)Reducing the harm of drinking and maintaining health

A moderate amount of biogenic amines can regulate blood pressure and promote cell growth, etc. Therefore, a small amount of alcohol consumption may have a positive effect on blood circulation and antioxidant, but each person's physical condition is different so it varies from person to person. Excessive amounts may cause negative symptoms such as headaches and vomiting, and may even cause cancer.

Regarding how to minimize the harm of alcohol consumption and maintain good health, Prof. Li suggests consumers to mitigate the potential harm by limiting the intake of foods with high biogenic amine content. The development of new products for our project could focus on reducing biogenic amine content and meeting customer needs.

(3) Questions about the amine-reducing brewer's yeast program

Regarding the process and principle of biogenic amine degradation by amine oxidase, Prof. Li said that the core of the process lies in the catalytic deamination of the amino groups in amines to produce ammonia and aldehydes. The aldehydes will then be further dehydrogenated to form acids, which will be gradually decomposed into carbon dioxide and water, ultimately leading to the complete degradation of biogenic amines.

Prof. Li gave the following insights on the advantages and suggestions of the project of "amine"-reducing Saccharomyces cerevisiae. The advantage lies in the fact that we have adopted the concept of synthetic biology to genetically modify the yeast, which is different from the previous method of screening strains that can degrade or not produce biogenic amines from nature, and provides a comprehensive solution to problems such as ensuring the quality of wines and degradation of biogenic amine content. and other problems such as ensuring wine quality and degrading biogenic amines. However, attention needs to be paid to adjusting the fermentation conditions, such as temperature and pH, to optimize the degradation efficiency of yeast in further studies.

3.3 Harvest and Prospect

Through the interview with Prof. Li Qi, we have gained a deeper understanding of the basics of biogenic amines, how to drink wine healthily, and how to reduce the "amine" of brewer's yeast.

(1) The three main factors affecting the production of biogenic amines are microorganisms, raw materials and fermentation conditions. Our project starts from microorganisms to degrade biogenic amines, and in the process of product development, we have to strictly control other conditions to ensure the safety of our products.

(2) A moderate amount of biogenic amines can have a positive effect on people's health, but an excessive amount can easily cause harm, in order to avoid the negative effects of biogenic amines, people should minimize the intake of fermented foods in their daily life.

(3) Regarding the suggestions to our project, Prof. Li affirmed the use of synthetic biology theory to degrade biogenic amines. Meanwhile we need to pay attention to the fermentation conditions in the experiment to achieve further optimization of the yeast degradation efficiency.

Figure 12:We are recording the results of the interview

4.1 Expert Introduction:

In order to understand the possible food safety issues that may arise when the new reduced "amine" yeast product is introduced to the market, we interviewed Prof. Qin Zhen from Shanghai University. Prof. Qin Zhen is a professor in the School of Food Science and Engineering at Shanghai University and is currently the Vice Dean of the School of Food Science and Engineering. His research is aimed at ensuring food safety, improving food quality and promoting the sustainable development of the food industry. He has rich research experience and outstanding academic achievements in the field of food science.

Figure 13：We are interviewing Dr. Qin

4.2 Interview Review:

(1) Food Production:

Biocontrol microorganisms are microorganisms that inhibit the growth of harmful microorganisms through interactions in the field of food safety. There may be food safety problems in the traditional alcohol brewing process. Low-level liquors such as wine and yellow wine may have problems with ethyl carbamate and pesticide residues. High-level wines including low-level wines also face the risk of methanol production from improper microbial control. Methanol is a toxic substance that can cause poisoning, blindness, and even death when ingested. Therefore, microbial control during the brewing process is of great food safety significance for all alcoholic beverages.

(2) Food safety norms:

Before putting the amine oxidase product into use, a series of tests need to be conducted. First, because the product uses genetically modified microorganisms, it needs to be declared to the Ministry of Agriculture to assess the safety of genetically modified microorganisms and conduct relevant approvals. Secondly, as a food additive, it needs to be declared as a new variety of food additives, including the demonstration of necessity and safety. Meanwhile, it needs to comply with the national standard GB2760. After passing these approvals, the product can be legally used in food. Regarding whether excessive intake of the product will have adverse effects on the body, this issue needs to be further evaluated in food safety experiments. In addition, as an enzyme preparation, the enzyme itself is a protein and overall does not have too many negative effects on the human body. However, what needs to be considered is whether the proteins in the product can cause allergic reactions and catalyze other negative reactions in the body or food.

(3) Amine oxidase applications:

Reducing biogenic amines in wine does not have an impact on shelf life. This is because according to national standards, alcoholic beverages with an alcohol content of more than 10% do not need to be labeled with a shelf life, and the longer time they are left out will not harm human health. Amine oxidase can not only reduce biogenic amines in alcohol but can also be applied to other foods. Amine oxidase can be used as a food additive during food processing to improve the biogenic amines in food. Not all foods that require fermentation are suitable for amine oxidase technology. This is because enzyme catalysis needs to comply with suitable pH and temperature conditions and requires a certain substrate concentration for conversion. High or low-temperature conditions and specific pH requirements may exist in food processing, and these factors may affect enzyme stability and reaction efficacy.

(4) Impact on the natural environment:

The impact of winemaking on the natural environment is studied in this study of enzymes used as food additives, where the production process involves genetically modified microorganisms and fermentation processes. During the production process, care needs to be taken to carry out biosafety controls to prevent the spread of genetically modified microorganisms into the environment. For alcohol companies using the enzyme product, they are purchasing a purified enzyme product that does not directly involve the upstream production process. However, the way the enzyme is applied can also have an impact on the winemaking process, depending on the steps in which the enzyme is added. If enzymes are added early in the fermentation process, they may have an impact on microbial growth and the fermentation process. In addition, the treatment of waste water, gas, and residues needs to be environmentally friendly.

Figure 14：We are asking questions about our topic.

4.3 Gain and Anticipation:

Through the interview with Prof. Qin Zhen, we gained a lot of information about our products in terms of food safety.

(1) Regarding the question on amine oxidase technology and food safety norms, we learned what kind of testing procedures need to be carried out for new products before they are actually put into food production, and from this, we learned more about the industry standards that we need to pay attention to in manufacturing our products. The answers from the experts allowed us to make our research more rigorous and our products better.

(2) Regarding the application of amine oxidase technology in winemaking, we have learned that the reduction of biogenic amines in fermented foods by means of amine-reducing yeasts does not affect the shelf life of the food in any way and that our product can be used in foods other than red wine, reflecting the fact that our product has a very promising future in terms of the market.

(3) With regard to the impact of minus "amine" yeast on the natural environment, it is known that this product, if added to food in an effective and healthy way, firstly, does not have any negative impact on the environment or on individuals, and secondly, it can be used in a wide range of food types.

Figure 15：We are recording the answers from Dr. Qin

5.1 Introduction

In order to understand the current development of wine brands and the potential of amine-reducing yeasts, we went on a field trip to Chateau Eiffel Changyu. Zhangyu is one of the earliest wine-making enterprises in China, founded in 1892. With a hundred years of wine-making heritage and the efforts of a professional team, Zhangyu Winery has won a good reputation and recognition in the domestic and international markets.

5.2 Zhangyu Aifei Chateau Field Trip

At Zhangyu Winery, we followed the lecturer to visit the layout of the whole campus as well as the factory workshop. In this field research, we learned the following points. First, wines can be classified according to characteristics such as sugar content, color, and the presence or absence of bubbles. Second, there are three practices of sparkling wine, the traditional method, the transfer method, and the tank method. Thirdly, the wine-tasting process mainly consists of three parts: color observation, smell, and taste.

Figure 16: We are in front of the winery.

Figure 17: The guide is introducing us the producing line.

5.3 Stakeholder Interview

(1) Wine Industry Pain Points：

There are several major pain points in the wine industry today. First, wine is over-taxed. Grapes are taxed according to the standard of economic crops and need to pay a 60% tax every year. As a result, domestic wine prices are relatively high. Second, China's mechanization development is relatively lagging behind, except for some large-scale production areas such as Xinjiang and Ningxia, other small production areas cannot afford the cost of mechanization. Thirdly, global climate change has a direct impact on grape yield and alcohol, and abnormal climate will also have a certain impact on the yield and quality of grapes, and consumers may not be able to accept such changes.

(2) Production technology for degraded biogenic amines：

At present, in winemaking, biogenic amines mainly come from the fermentation process, and reasonable control of the fermentation environment can reduce the possibility of biogenic amine production, so as to keep their content low. Nowadays, there is not much demand for biogenic amine degradation technology in the domestic market, but with technological advancement, including the development in indigenous yeast R&D and multi-yeast fermentation, the demand for biogenic amine degradation technology will gradually increase. Our topic of degrading biogenic amines using amine oxidase based on synthetic biology is a very promising project.

(3) Marketing Approach：

The winery's current promotional methods include both online and offline channels. Online is mainly promoted by the company's specialized media and branding departments. For offline promotion, the Zhangyu brand mainly adopts traditional marketing methods, such as participating in booth exhibitions and organizing events such as wine tastings. For the winery, it focuses on one of its high-end products, which include specialized wines such as Cabernet, and also produces ordinary wines. From the producer's point of view, the brand is categorized according to different production lines and product portfolios to meet the needs of different consumer groups.

5.4 Summarize：

Through the interviews and tours of the Zhangyu winery, we learned about the current market situation of the winemaking industry and the market prospects that our product will face after its launch. The interviews helped us to better understand the market of the winemaking industry so that we can further improve our business plan.

Figure 18: We are visiting the winery.

Figure 19: We are visiting the grape garden.

The questionnaire research provided insights into the target audience, market potential, price range, and purchase tendencies for the future product.

The interview with Professor Li Qi highlighted the importance of ensuring product safety during development, avoiding negative impacts of biogenic amines, and optimizing yeast degradation efficiency through careful fermentation conditions.

The interview with Professor Qin zhen underscored the significance of adhering to industry standards in the testing procedures before the production of the new product.

The field trip to Chateau Eiffel Changyu provided insights into the current wine industry market and post-launch prospects, enhancing our business plan.

l Our target users are wine producers, and our goal is to establish partnerships with them to aid in reducing biogenic amine content, enhancing food safety, and improving product quality.

l We have harnessed genetic engineering techniques to introduce the Aocl (amine oxidase, copper-containing) and FAD (flavin adenine dinucleotide) genes into yeast cells, equipping them with the ability to break down biogenic amines. This collaboration aims to encourage the adoption of amine-reduced yeast, a significant yeast cell modification with practical importance. This technology is particularly valuable in environments or industries where biogenic amine levels are high, such as food fermentation processes.

l In the real world, we'll start with a cost-effective promotion budget, ensuring steady profits and reinvesting in sustained advertising. We'll utilize various media platforms, seek endorsements from experts and authoritative organizations, and establish partnerships with wine producers to promote amine-reduced yeast adoption.