Study for enhancing γ-aminobutyric acid (GABA), sialic acid, and immune response in fermented deer antler velvet using lactic acid bacteria strains = 젖산균주를 이용한 녹용발효액의 감마아미노부티르산(GABA), 시알산 및 면역반응 증진에 관한 연구

다국어 초록 (Multilingual Abstract)

Deer antler velvets are widely used in traditional medicine for their anti-aging, antioxidant, and immunity-enhancing effects. Although there are studies on deer antler velvet extract (AV) fermented using Bacillus subtilis, very little is known about the effects of AV fermented with probiotic stains. This study aims to evaluate whether each probiotic strain selected for fermented antler velvet (FAV) has distinctive characteristics and produces unique changes to the metabolic profiles of FAV during fermentation as well as immune-enhancing activity. All the strains exhibited high proliferation and survival with 108–109 CFU/g in FAV. Two probiotic strains (L. rhamnosus LFR20-004 and L. sakei LFR20-007) were selected based on their results that they significantly improved sialic acid contents, γ-aminobutyric acid (GABA) contents of AV and extended Caenorhabditis elegans life span. 65 metabolites were detected by gas chromatography-mass spectrometry (GC–MS) in the FAV. FAV with L. rhamnosus LFR20-004 (FAV-4) is predicted to be able to synthesize 5 amino acids (aspartic acid, glycine, proline, serine, and valine), including essential amino acids. Also, co-fermentation with both strains (FAV-Co) increased the concentrations of 5 organic acids (citramalic acid, lactic acid, malic acid, malonic acid, and succinic acid). FAV not only improves the lifespan of C. elegans, but also extended the survival of C. elegans infected with Salmonella typhi, Listeria monocytogenes, and E. coli O157. Exposure of Cyclophosphamide (Cy) to mice splenocytes reduced cell viability, but the treatment of FVA increased cell viability compared to Cy only treated cells. Interestingly, cytokines such as IL-6, IL-10, TNF-α, IFN-γ were significantly increased in spleen tissues of the Cy-induced immunosuppressed mice. These results showed that FAV might stimulate the immune reaction and provide the scientific basis for dietary supplementary as probiotics-fermentation of deer antler velvet.

국문 초록 (Abstract)

녹용은 노화 방지, 항산화 및 면역 강화 효과로 전통 의학에서 널리 사용된다. Bacillus subtilis를 이용하여 발효시킨 녹용 추출물에 대한 연구는 있으나 프로바이오틱스로 발효시킨 녹용의 효과에 대해서는 알려진 바가 거의 없다. 본 연구는 발효 녹용(FAV)을 위해 선별된 각 프로바이오틱스 균주가 독특한 특성을 가지고 발효 중 FAV의 대사 프로파일에 고유한 변화를 일으키는 지와 면역 강화 활성을 평가하는 것을 목표로 한다. 모든 균주는 FAV에서 108-109 CFU/g로 높은 증식 및 생존율을 보였다. 2개의 프로바이오틱 균주 (L. rhamnosus LFR20-004 및 L. sakei LFR20-007)는 이들 균주가 발효 후 녹용 추출물의 시알산 및 γ-아미노부티르산(GABA) 함량을 모두 증가시키고 C. elegans의 수명을 유의적으로 개선했다는 데이터를 기반으로 선택되었다. 65개의 대사산물이 FAV에서 가스 크로마토그래피-질량 분석법(GC-MS)에 의해 검출되었다. L. rhamnosus LFR20-004 (FAV 4) 를 포함하는 FAV는 필수 아미노산을 포함하여 5개의 아미노산 (아스파르트산, 글리신, 프롤린, 세린 및 발린) 을 합성할 수 있을 것으로 예측된다. 또한, 두 균주 (FAV-Co) 와의 동시 발효는 5가지 유기산 (시트라말산, 젖산, 사과산, 말론산 및 숙신산)의 농도를 증가시켰다. FAV는 C. elegans의 수명을 향상시킬 뿐만 아니라 Salmonella typhi, Listeria monocytogenes 및 E. coli O157에 감염된 C. elegans의 생존 기간을 연장했다. Cyclophosphamide (Cy)를 생쥐 비장 세포에 노출시키면 세포 생존력이 감소했지만 FVA 처리는 Cy만 처리한 세포에 비해 세포 생존력을 증가시켰다. 흥미롭게도 IL-6, IL-10, TNF-α, IFN-γ와 같은 사이토카인은 Cy 유도 면역억제 마우스의 비장 조직에서 유의하게 증가하였다. 이러한 결과는 FAV가 녹용의 프로바이오틱스 발효로서 면역 반응을 자극하고 식이 보충제의 과학적 근거를 제공할 수 있음을 보여주었다.

목차 (Table of Contents)

• Chapter 1. Literature review 6
• 1.1 Deer antler velvet 6
• 1.1.1 Uses and efficacy of deer antler velvet 6
• 1.1.2 Chemical constituents of deer antler velvet 7
• 1.1.3 Pharmacological properties of deer antler velvet 7
• 1.1.4 Research for domestically deer antler velvet 8
• 1.2 Probiotics 13
• 1.2.1 Definitions of probiotics and health-promoting effects. 13
• 1.2.2 Fermented foods by probiotics. 14
• 1.2.3 Probiotics and immunity 14
• 1.3 Caenorhabditis elegans 16
• 1.3.1 Caenorhabditis elegans as a model system to study prolongevity. 16
• 1.3.2 Description of the main pathways 17
• 1.4 Cyclophosphamide-induced immunosuppressed mice 19
• 1.4.1 Immune promoting effects 19
• 1.4.2 Cyclophosphamide 20
• Chapter 2. Material and methods 23
• 2.1 Bacterial strains 23
• 2.2 Bile and acid tolerance 23
• 2.3 C. elegans longevity assay 24
• 2.4 Antler velvet extract fermentation 24
• 2.5 Total acidity, pH, and bacterial growth 25
• 2.6 Total nitrogen, amino acid, and protein quantification 25
• 2.7 Gamma-aminobutyric acid analysis 26
• 2.8 Sialic acid analysis 27
• 2.9 Metabolite analysis 27
• 2.10 C. elegans killing assay 29
• 2.11 Isolation of mouse splenocytes 29
• 2.12 Measurement of cell cytotoxicity and proliferation 30
• 2.13 Cy-induced immunosuppression mouse model 30
• 2.14 RNA isolation and quantitative real-time reverse-transcription-polymerase chain reaction (RT-qPCR) 31
• 2.15 Statistical analyses 31
• Chapter 3. Results and discussion 33
• 3.1 Resistance of LAB strains to artificial stomach conditions 33
• 3.2 The ability of LAB strains to adhere to epithelial cells 36
• 3.3 Probiotic candidates for fermentation 38
• 3.4 Selection of LAB strains using C. elegans 41
• 3.5 Proteolysis in fermented antler velvet 43
• 3.6 GABA contents 45
• 3.7 Sialic acid contents 47
• 3.8 Select optimal strains 49
• 3.9 Comparison of Volatile Metabolomic Profiles 51
• 3.10 Metabolite changes in four sample types 54
• 3.11 C.elegans life span 58
• 3.12 GFP in C.elegans 60
• 3.13 Effects and mechanisms of prolongevity induced by FAV in C. elegans 62
• 3.14 Effect of FAV extract on C. elegans infected with pathogens 64
• 3.15 Cytoprotective effect of FAV on mouse splenocytes 67
• 3.16 Conclusion 71
• Reference 72
• Summary in Korean 88
• Acknowledgment ························································································································ 90