Shoshi Inooka*

doi:10.19080/ARR.2025.12.555839

Research Article

Separation Of Antibiotic-Producing Cells from Beer Produced in Co-Cultures of DNA (Streptomyces) Crown Cells with Yeast

Shoshi Inooka*

Japan Association of Science Specialists

Submission:January 28, 2025; Published:February 04, 2025

*Corresponding author:Shoshi Inooka, Japan Association of Science Specialists

How to cite this article:Shoshi I. Separation Of Antibiotic-Producing Cells from Beer Produced in Co-Cultures of DNA (Streptomyces) Crown Cells with Yeast. Paper Recycling Ann Rev Resear. 2025; 12(3): 555839.DOI: 10.19080/ARR.2025.12.555839

Abstract

DNA crown cells (artificial cells), which can proliferate within egg white in vivo, can be prepared using sphingosine (Sph)-DNA-adenosine-monolaurin compounds. In a previous experiment, antibiotics were produced in co-cultures of yeast and DNA (Streptomyces) crown cells during the production of beer. However, the mechanism of antibiotic production remained unclear. The present experiments examined whether antibiotic-producing cells could be separated from the previously produced beer. Here, it was described that antibiotic-producing cells (antibiotic Crown Strep-cells) were separated using white egg and monolaurin from the previously produced beer.

Keywords:DNA (Streptomyces) crown cells; Sphingosine-DNA; Antibiotic Crown Strep-cells; Monolaurin, Beer

Introduction

Artificial cells have potentially useful applications in the study of a wide variety of problems in life sciences. However, until the first report by the present author in 2012 [1], fully functional (self-replicating) cells had not been produced. In 2016, the principal methods for generating artificial cells capable of multiplication within egg white and cultivation in vitro were reported [2]. In the same year, artificial cells referred to as DNA crown cells were reported by the present author [3]. The cells derive their name from their exterior structure, which consists of DNA. DNA crown cells were synthesized using commercially available compounds: sphingosine (Sph), DNA, adenosine, and monolaurin. These cells were shown to generate various types of structures and exhibit cell proliferation when treated with monolaurin or egg white [4–7]. Additionally, it was demonstrated that antibiotics were produced during the co-culture of DNA crown cells with yeast during the production of beer, suggesting that the DNA crown cells were involved in antibiotic production [8–11]. However, the mechanisms underlying antibiotic production remained unclear because antibiotic-producing cells could not be successfully separated. The present study examined whether antibiotic-producing cells could be separated from antibiotic-containing beer produced in 2018 through co-cultures of DNA (Streptomyces) crown cells with yeast, which had been stored at room temperature [10]. This report describes the successful separation of antibiotic-producing cells from such beer.

Materials and Methods

Materials

Antibiotic-containing beer: Beer produced in 2018 was used in this study. The methods for producing this beer have been reported previously [8]. However, for clarity and to facilitate understanding of the present experiments, these methods are described briefly below. The materials used were the same as those employed in previous studies [12,13]: Sph (Tokyo Kasei, Tokyo, Japan), DNA extracted from Streptomyces, adenosine (Sigma-Aldrich, USA, Wako, Japan), monolaurin (Tokyo Kasei), and adenosine-monolaurin (A-M), a compound synthesized from a mixture of adenosine and monolaurin [12,13]. Monolaurin solutions were prepared to a final concentration of 0.1 M in distilled water. Agar plates were prepared using standard agar medium (SMA) (AS ONE, Japan). Egg white was obtained from a local market. Materials for beer preparation and antibiotic testing were: Malt (Black Roch PILSENER, New Zealand); Yeast (Dry ale yeast, Safale S-04, Formentis, Bergues, France); Potato dextrose agar (PDA) (Kyodo Nyugiou, Tokyo, Japan); and Bacillus subtilis natto (Daikokuya, Nagoya, Japan).

Methods

Preparation of beer containing antibiotics: We first prepared DNA (Streptomyces) crown cells [8]. Briefly, 180 μL of Sph (10 mM) and 50 μL of DNA (1.7 μg/μL) were combined, and the mixture was heated and cooled twice. Then, A-M solution (50 μL) was added. The resulting suspension was used to inoculate egg white and incubated for 7 days at 37°C. Then, egg white was collected and used as the DNA (Streptomyces) crown cells.

Preparation of beer

1) Approximately 3 g of dry yeast was mixed with 15 mL of egg white and incubated for 5 h at 37°C.
2) Then, 10 L of beer malt was added to the egg white with yeast and incubated for 18 days at room temperature.
3) Ａportion of the old malt was removed and 5 L of fresh malt was added.
4) After approximately 4 weeks of incubation at room temperature, a portion of the old malt removed, and 5 L of fresh malt was added.
5) Beer containing antibiotics could be prepared after approximately 5 weeks of culture.
a) The antibiotic assay of the prepared beer (400 μL) is shown in Figure 1.
b) Clear zones observed on the plates indicated antibiotic activity.

Cell separation from beer using agar plates

1) A 100 μL sample of beer was poured onto three agar plates and incubated for 2 days at 37°C.
2) Approximately 1.5 mL of egg white was poured onto the plates.
3) The plates were incubated for 2 days at 37°C.
4) Approximately 1.5 mL of 0.1 M monolaurin was poured onto a plate.
5) The plates were then incubated for a further 2 days at 37°C.

Cultivation of separated cells in malt

1) Samples from the plates were picked and transferred to microtubes containing 1 mL of malt supplemented with 10 mg of dry yeast. The tubes were incubated for 3 days at 37°C.
2) The resulting cultures were tested for the presence of antibiotics.

Cell separation from microtube culture fluids and antibiotic assay of cells
i. A 100 μL of microtube culture fluid was poured onto agar plates and incubated at 37°C for 24 hours.
ii. Growing bacterial colonies were picked and transferred directly to 5 mL of malt. The cultures were incubated for 5 days at room temperature.
iii. The culture fluid was subsequently tested for antibiotic production.

Preparation of plates for antibiotic production assay: The antibiotic production assay was conducted using the agar well diffusion method, as described previously [9]. The test bacteria were mixed with 200 mL agar medium and poured into Petri dishes. A well of approximately 2 cm in diameter was created on each plate. Approximately 400 μL of sample was added to each well and the plate was incubated for 18 h at 37°C. After incubation, the presence or absence of inhibition zones was recorded to determine antibiotic activity.

General observations: Objects on plates were observed directly with the naked eye.

Results

Figure 2 shows a photograph of an agar plate containing beer after 2 days of white egg culture. No objects visible to the naked eye were observed on the Petri dish, which had a diameter of 8.0 cm. Figure 3 shows a photograph of an agar plate after 2 days of culture following monolaurin treatment after egg white treatment. Cloudy, dot-like or round objects visible to the naked eye were observed on the Petri dish, which had a diameter of 8.0 cm. Figure 4 shows a photograph of a plate from an antibiotic test of malt fluids after 3 days of incubation with separated cells. A clear zone was observed indicating antibiotic activity. Figure 5 shows a photograph of an agar plate culture of malt fluids from Figure 4. Many colonies were observed after 1 day without monolaurin or egg white treatment. Figure 6 shows a photograph of an agar plate from an antibiotic test of malt culture fluids containing objects from Figure 5. A clear zone was observed indicating antibiotic activity. In previous studies [8–10], it was reported that antibiotics were produced in co-cultures of DNA (Streptomyces) crown cells with yeast. However, the mechanisms of antibiotic production remained unclear because antibiotic-producing cells could not be separated. In the present experiments, it was demonstrated that antibiotic-producing cells were successfully separated from beer produced in 2018. This antibiotic-containing beer was obtained after approximately 5 weeks of co-culture. However, no yeast or viable cells grew when the beer was cultured on agar medium at 37°C or at room temperature (approximately 20°C). Similarly, no objects were observed when the beer was used for cultivation on an agar plate. Following treatment with egg white and monolaurin, cells were observed to grow. When these cells were cultured with yeast in malt at room temperature, antibiotic activity was detected.

In contrast, in previous experiments [4–7], when synthetic DNA (E. coli, Human placenta/Ascidian, Streptomyces) crown cells were treated with egg white or monolaurin on agar medium, cell proliferation or objects similar in appearance to double cells, morning glories, daffodils, and cell rings were observed. These findings suggest that the growth of DNA crown cells may be influenced by the presence of egg white or monolaurin, and such cells may be separated from the beer. Furthermore, when the proportion of DNA crown cells was high relative to yeast during beer production, antibiotics were detected after 3 days. This observation suggests that the growth of antibiotic-producing cells may depend on DNA crown cells [11]. However, the mechanism by which such antibiotic-producing cells arise remains unclear. The occurrence of these cells could be attributed to either scientific or non-scientific factors. From a non-scientific perspective, the origin of the cells may be linked to the materials used in the process (e.g., dry yeast or malt), or due to contamination from the surrounding environment.

From a scientific perspective, the origin of the cells may be attributed to DNA crown cells or DNA crown cells influenced by yeast. It is also possible that the cells are neither yeast nor yeast influenced by DNA crown cells, such as through gene transfer. This is because the antibiotic-producing cells independently produce antibiotics without any contribution from yeast. However, the precise origin remains unclear. Further experiments are necessary to establish the origin of these cells and to clarify their characteristics, including their morphology and antibioticproducing capabilities (e.g., whether the cells can continuously produce antibiotics indefinitely). In conclusion, antibiotic production was observed in co-cultures of DNA (Streptomyces) crown cells with yeast. The present study also demonstrated that antibiotic-producing cells could be separated from beer through treatment with egg white and monolaurin. Various DNA crown cells may proliferate in response to these treatments, as described previously [14-16].

These findings suggest the potential to separate additional antibiotic-producing cells using egg white or monolaurin treatment. To avoid confusion, the identified cells were named as follows: Antibio Crown Strep cells B-1: Antibiotic-producing DNA (Streptomyces) crown cells derived from beer. Streptomyces in parentheses denotes the DNA source, and “B” refers to beer as the source of the cells (The author appreciate Microsoft Copilot for its’ assistance in naming). It is also important to clarify whether the produced antibiotic has clinical value and to investigate whether its production is associated with drug (antibiotic) resistance or drug-resistant bacteria. Future studies will focus on whether additional antibiotic-producing cells can be separated using the described methods (egg white or monolaurin stimulation) and whether these antibiotics have practical applications.