Drinking is a recreational activity for people. People can make friends and even find a soulmate at an after-work drink or a party. But traditional liquor has an indelible problem: it has lots of biogenic amine. Biogenic amines are substances that include nitrogen. Biogenic amines can make people get headaches and stomachaches and also nervous.[1] The available technology can decrease the concentration of biogenic amines but will influence the flavor.[2] We want to find if any method can keep the taste and have a low concentration of biogenic amines. That’s the reason why we chose this topic.

Now, the primary method of biodegradation of amines is to extract the amine oxidase secreted by microorganisms and catalyze the oxidation of amines to crack them into aldehydes and ammonia. It was found that the amine oxidase extract of Penicillium citricum could degrade the concentration of biogenic amines in red wine, and no significant changes in phenolic substances and volatile components were observed in wine, which did not affect the product's sensory properties.[3] However, compared with isolating and purifying microbial amine oxidase, using strains that can directly degrade amines is more convenient. Bacteria with active amine oxidase can degrade biogenic amines in food and be used as a starter for some fermented foods.[4] Lactic acid bacteria can reduce biogenic amines in fermented wine, and Lactobacillus and pentococcus have the best degradation effect.[5] Lactobacillus casei IFI-CA 52 has a good application prospect as a fermentation strain to reduce biogenic amines in wine.

 

We separated the experiment into three parts. The first part is the combination of recombination plasmid. The second part is yeast conversion and protein expression. The last part is function testing.

In the first part, we will use pAO815(vector), AocⅠ(gene), and FAD(gene). AocⅠ is an ammonia oxidase that catalyzes and splits the biogenic amine. FAD can help the plasmid expression better. The above three genes were prepared and mixed by centrifuge. Then heating, 95° C-5min, unspin 95° C-30s; Annealing 55°C-30; Extending 72°C -2min30s (at a rate of 1000bp per minute, depending on the replication target length). Those three steps are a cycle we need to amplify 30 times. After that, we need to check the function. So, we need to use agarose gel electrophoresis. We must also make a culture medium for LB and yeast while recombining plasmid (pAO815-AocⅠ and pAO815-AocⅠ -FAD). At last, we do the functional test --- transparent circle experiment.

 

Our terminal goal is to make a healthier liquor. To achieve this, we need to create a new yeast with a gene editing technique within our recombinant plasmid pAO815-AocⅠ-FAD in order to express the enzyme for amine degradation, therefore the liquor fermented by our yeast could have a lower amine level but the same taste quality.

Besides, in the future, Biogenic amine reduction in Saccharomyces cerevisiae will be extended to other fermented foods with high biogenic amines, such as canned fish products, fermented sausage, and cheese, to broaden its application scope further. The next step is to investigate amine oxidase's biological activity and biogenic amine's degradation mechanism to provide a theoretical basis for applying amine oxidase.

 

[1] Shalaby, A. S. G. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29(7), 675–690.

[2] Benkerroum, N. (2016a). Biogenic amines in dairy products: origin, incidence, and control means. Comprehensive Reviews in Food Science and Food Safety, 15(4), 801–826. 

[3] Niu, T., Li, X., Guo, Y., & Ma, Y. (2019). Identification of a Lactic Acid Bacteria to Degrade Biogenic Amines in Chinese Rice Wine and Its Enzymatic Mechanism. Foods (Basel, Switzerland), 8(8), 312. https://doi.org/10.3390/foods8080312

[4] Ruiz-Capillas, C., & Jiménez-Colmenero, F. (2005). Biogenic amines in meat and meat products. Critical Reviews in Food Science and Nutrition, 44(7-8), 489-599.

[5] Lonvaud-Funel, A. (2001). Biogenic amines in wines: role of lactic acid bacteria. Fems Microbiology Letters, 199(1), 9–13.