Proof of Concept

1.1 Product outline

Product (non-proprietary) common name: Ø-HAS Saccharomyces cerevisiae strain series.

Strain size: 10g/pack.

Product dosage form: bagged yeast powder.

Color: White or pale yellow.

Ingredient: Strain Extract.

Storage conditions: -80°C in Minus 80 Freezer.

Shelf life: about 20 months at -80°C.

1.2 Product utilize

This series of strains can be used by brewing companies to manufacture alcoholic beverages with low yields of higher alcohols. The alcoholic beverages produced by this strain can be consumed by people in all countries and regions (provided the drinkers comply with the laws and regulations of their corresponding regions or countries and no relevant medical history).

1.3 Precautions for use

For brewing companies:

1. Please transport and store yeast at the specified temperature.
2. Please do not expose the strain anywhere other than in a fermenter or sterile environment.
3. Please use strains within the shelf life.
4. After the beverage is produced, please do a good job in quality monitoring.
5. Please sell alcohol products within the shelf life and comply with relevant regional laws and regulations.

For drinkers:

1. Before drinking, please pay attention to your drinking behavior is legal in the relevant region or country.
2. Be aware if you have a chronic medical condition that can be affected by alcohol or have a history of alcohol-related medical conditions (e.g., alcoholism, liver failure)
3. Please drink in moderation.
4. Please note that alcoholic beverages are within their expiry date.
5. Please refrain from behaviors that endanger social stability after drinking alcohol.

Please stop using it when the following conditions occur:

For brewing companies:

1. Use contaminated strains for alcoholic fermentation (e.g., if the packaging is broken).
2. The product has abnormal detection values.
3. Does not comply with local regulations.
4. Not enough production conditions.
5. Use this strain for relevant human experiments.

For drinkers:

1. Taking drugs that are contraindicated for alcohol. (e.g., hypnotics, antihypertensive drugs, hypoglycemic drugs, antibiotics, antidepressants)，that it can produce such as disulfiram reaction or other life-threatening reaction.
2. Drink alcohol in violation of relevant regional laws and regulations (e.g., drive).
3. Life-threatening symptoms such as facial flushing, blurred vision, throbbing headache, dizziness, nausea, vomiting, sweating, chest pain, myocardial infarction, dyspnea, and convulsions occur.
4. Has history of symptoms such as the third related symptoms after drinking alcohol.
5. Acts that endanger the peace and stability of society after drinking alcohol.

Warning:

1. Taking into account the congenital or acquired influence, it cannot be ruled out that there will be unpredictable adverse reactions.
2. Minors are prohibited from drinking alcohol.
3. The alcoholic beverages produced by this strain can only be consumed and cannot be used for other purposes.

2.1 Brewing enterprise

Once the brewing business has purchased our strains, we will deliver them in a safe and fast manner.

The brewing enterprise should unpack the bagged strains in the sterile brewing workshop, put it into the fermentation tank of the alcohol fermentation environment, and decide the fermentation time, system and environment according to the enterprise's own needs. When the brewing is complete, the company will send the product to a supermarket or other sales platform according to their alcoholic beverage storage conditions.

During the brewing process, the BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R homologous gene fragment that we replaced inhibits the production of higher alcohols, which are derived from the replaced BAT2 produced by genes. This can effectively reduce the content of higher alcohols in alcoholic beverages.

If the enterprise does not need to use the strain within 2h, please put the strain in Minus 80 Freezer.

2.2 Drinkers

If the brewing company is honest, please feel free to drink the fermented products of our strains.

3. Feasibility testing and analysis

After confirming that our desired homologous gene fragment BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R was successfully constructed (Figure 3-1 is the gel electrophoresis result of this fragment), we It was introduced into Saccharomyces cerevisiae strain template using lithium acetate transformation method, and a series of tests were carried out to prove whether the strain achieved our experimental goal.

Fig 3-1 shows the gel electrophoresis result of gene fragment BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R.

To test for its functional integrity, we performed a growth curve test on this strain.

When the results were obtained, we performed data modeling to see if the modification had an effect on the growth of the strain, for which we made an applied mathematical model to study the relationship between the absorbance of OD₆₀₀ and the growth time of strain A and the wild-type strain. Then, we predicted the maximum OD₆₀₀ absorbance value of the strain according to the model results, which was the maximum growth amount. We use the following models for experiments:

f(x) = a / (b + exp(-c ∙ x))

Figure 3-2 and 3-3 is the mathematical model of Strain A and wild type strain.

Fig 3-2 shows the mathematical model of wild type strain;
Fig 3-3 shows the mathematical model of Strain A.

The modeling results showed that the OD₆₀₀ absorbance of strain A and wild-type strain increased first and then tended to be stable with time. In addition, the OD₆₀₀ absorbance of strain A was higher than that of the wild-type strain, indicating that the genetic modification did not affect the growth of strain A compared with the wild-type strain.

Furthermore, we also conducted a fermentation test to simulate the brewing alcohol environment to prove that the strains we constructed could successfully inhibit the production of higher alcohols.

We did a ration of hours of fermentation. After the fermentation was completed, we used a high-performance liquid chromatograph to detect the content of various alcohols to form the gas phase peak maps.

Figure 3-4 to 3-7 shows the gas phase peak maps of Strain A and Wild-type strain.

Fig 3-4 and 3-5 shows the gas phase peak map of Wild-type strain.
Fig 3-6 and 3-7 shows the gas phase peak map of Strain A.

According to the experimental data, it is organized as the following table 3-1:

Table 3-1 shows the data of different types of alcohol of Strain A and Wild-type strain.

Tests have shown that the strain we constructed reduces the higher alcohols on the drink by 152.6 mg per liter, which is 38% of the wild type strain, and the alcohol production is reduced by 0.15% vol which is 87.234% of the wild type strain. This means that our strains have successfully and effectively reduced the production of higher alcohols during the brewing process. In a nutshell, we show that it works, it is viability, of our concept.