開發具延緩衰老及抗氧化功效之發酵乳產品

Abstract

隨著科技進步人類壽命延長，全球老年人口比例逐年增加，根據臺灣行政院主計總處統計，2022 年底，65 歲以上人口占總人口 17.56%，共計超過 400 萬人，人口比例已高於 14 歲以下人口，根據國家發展委員會預估，2025 年，老年人口比例將突破 20%，即進入超高齡社會。人口高齡化是全球未來的趨勢，各國的國家衛生及社會系統面臨險峻的挑戰，老年人的疾病預防及照顧至關重要，近期腸腦軸的研究揭示了中樞神經系統和腸道之間雙向溝通的關係，腸道菌相失衡可能破壞腸道屏障，使腸道通透性增加及誘發腸道免疫反應，進而引起全身性的慢性發炎，最終導致許多慢性疾病，如：肥胖症、高血壓、糖尿病、心血管疾病、慢性腎臟病等，其中也包含許多與衰老相關的疾病，在某些情況下，血腦屏障被破壞，引起神經發炎、神經損傷和神經元損失，最終導致神經退化性疾病。先前的研究發現，Lactobacillus kefiranofaciens HL1 具有強大的抗氧化能力和延緩衰老之功效。因此，本研究的目標是以 Lb. kefiranofaciens HL1 作為發酵菌株，開發出一種新穎的發酵乳產品，並以 D-galactose 誘導 BALB/c 小鼠老化模式評估其抗氧化能力、抗發炎和延緩衰老之功效，並探討其可能的作用機制及腸腦軸之間的關聯。

本研究分為兩個部分。第一部分目標是開發出以 Lb. kefiranofaciens HL1 發酵而成之發酵乳製品，並測試其物理特性、保存條件及官能品評。首先，由於 Lb.kefiranofaciens HL1 單株益生菌在 30°C 下無法於 24 小時內完成發酵，因此研究添加 Lactococcus lactis subsp. cremoris APL015 (因為其生長快速，且具有良好的產酸及產胞外多醣的能力)，進行共同發酵，以降低發酵時間，並開發出兩款發酵乳產品，FM 發酵乳為 Lb. kefiranofaciens HL1 及 Lc. lactis subsp. cremoris APL015 共同發酵而成的發酵乳產品，FS 發酵乳作為對照組，為 Lc. lactis subsp. cremoris APL015單株發酵而成之發酵乳產品。結果顯示，FS 單株發酵乳及 FM 共同發酵乳均能在16 小時內使 pH 值下降到 4.6 以下，達到發酵終點，並達到 9 log CFU/mL 之活菌數。在 4℃下及剪切率 30 s-1 下，FS 和 FM 組的表觀黏度均高於以商業優格菌株(保加利亞乳桿菌及嗜熱鏈球菌) YC-380 發酵而成的 Yogurt 組，且離水率顯著低於Yogurt 組，此外，FS 和 FM 組之堅實度及濃稠度皆顯著高於 Yogurt 組，顯示 FS及 FM 發酵乳相較於商業優格菌株發酵而成之發酵乳具有更穩定的膠體結構，有利於加工製成及減少食品添加物的使用。然而，在我們測量的任何物理特性 (離水率、表觀黏度、堅實度、濃稠度、黏聚性及內聚功) 中，FS 組和 FM 組之間皆沒有顯著差異，這表示添加 Lb. kefiranofaciens HL1 共同發酵並不會改變 Lc. lactis subsp. cremoris APL015 之發酵特性。

在第二部分中，我們使用 D-galactose 誘導 BALB/c 老化小鼠模式來評估發酵乳的抗氧化能力、抗發炎和延緩衰老之功效。實驗將 60 隻小鼠分為 6 組，分別為健康組 (positive control, PC)、D-galactose 誘導老化組 (negative control, NC)、餵飼單一菌株組 (Lb. kefiranofaciens HL1, HL1)、餵飼單一菌株組 (Lc. lactis subsp. cremoris APL015, APL)、餵飼 FS 單株發酵乳組 (FS)、餵飼 FM 共同發酵乳組 (FM)進行功效評估。首先，行為試驗方面，在莫里斯水迷宮實驗中，HL1 組於訓練第 3天及第 4 天之脫逃時間皆顯著低於 NC 組，而在 Y 字迷宮自發性探索實驗中 PC 組及 FM 組相較於 NC 組有更高的交替率，結果顯示給予 HL1 單菌株之菌液能顯著的改善小鼠之學習力及長期空間記憶能力，而給予 FM 共同發酵乳則對於改善小鼠之短期空間記憶能力有較顯著的效果。在分子層級上，小鼠犧牲後測量血清、肝臟及大腦中之介白素-1β(IL-1β)及腫瘤壞死因子-α (TNF-α) 等促發炎細胞因子來評估各器官之發炎程度，抗氧化酵素如超氧化物歧化酶 (SOD) 之活性以及脂質過氧化生成的丙二醛 (MDA) 濃度來評估氧化壓力，我們觀察到 HL1 組與 NC 組相比，能夠顯著降低血清中 TNF-α 及 IL-1β、肝臟中 TNF-α、大腦中 TNF-α 及 MDA的濃度，並顯著增加大腦中 SOD 的濃度。此外，FM 組能夠顯著降低血清中 IL-1β、肝臟中 TNF-α、大腦中 TNF-α 及 MDA 的濃度，並顯著增加大腦中 SOD 以及在盲腸內容物中所含的丁酸濃度。最後，我們使用 qPCR 分析了盲腸微生物菌相組成的變化。與 NC 組相比，FM 組能顯著降低產氣莢膜梭菌的比例。

綜上所述，我們以 Lb. kefiranofaciens HL1 和 Lc. lactis subsp. cremoris APL015開發出了一種新穎的發酵的乳製品，具有良好的質地與加工特性，口感一致，且在運輸和儲存過程中具有良好的穩定性。這種發酵乳製品還具有良好的抗氧化、抗發炎和抗衰老特性。

By the end of 2022, the elderly population, aged over 65 years old, accounted for 17.56% of the total population in Taiwan. It is now generally accepted that chronic systemic inflammation and neuroinflammation play important roles in aging process and the onset of neurodegenerative disease. Recently, the gut-brain axis revealed the bidirectional communication between the central nervous system and the gastrointestinal tract. Dysbiosis in the gut microbiota might resulted in various chronic diseases via upregulating gut barrier permeability and activating immune reaction with systemic inflammation. In severe cases, the breakdown of the blood-brain barrier evoke neuroinflammation, neural injury, and neuronal loss, culminating in neurodegenerative disorders. Previous research has identified Lactobacillus kefiranofaciens HL1, isolated from kefir grains as a probiotic strain with great anti-oxidative properties and anti-aging effects both in vitro and in vivo. Consequently, the objective of this study is to develop a novel fermented milk product containing Lb. kefiranofaciens HL1 and estimate its anti-oxidative, anti-inflammatory, and anti-aging effects in D-galactose-induced aging mice model.

The experiment was divided into two main phases. Initially, efforts focused on the development of fermented milk products and investigate their physical properties. Since the fermented milk solely using Lb. kefiranofaciens HL1 strain failed to reach the fermentation endpoint within 24 hours, we developed a fermented milk product (FM) by mixing it with Lactococcus. lactis subsp. cremoris APL015, which has strong acid producing ability to reduce fermentation time. Additionally, a control group comprising another fermented milk product (FS), fermented soley with Lc. lactis subsp. cremoris APL015, was established. Results indicated that both FS and FM achieved the target pH of 4.6 and achieve 9 log CFU/mL in the counts of LAB strains within 16 hours. In the texture analysis, both FS and FM groups showed lower water syneresis and higher apparent viscosity (at 4°C and a constant shear rate of 1 30 s-1) than the Yogurt group, which was fermented by the commercial yogurt culture YC-380. Furthermore, FS and FM groups also exhibited higher consistency and firmness compared to the Yogurt group. However, there’s no significant difference between FS and FM groups in any physical property we measured (water syneresis, apparent viscosity, firmness, cohesiveness, work of cohesion, and consistency).

In the second phase, a D-galactose-induced aging mouse model, utilizing BALB/c mice, was employed to assess the anti-oxidative, anti-inflammatory and anti-aging effects of the fermented milk products. Sixty mice were divided into six groups: a healthy group (PC), an induced aging group (NC), groups orally administrated with single strains of Lc. lactis subsp. cremoris APL015 (APL) or Lb. kefiranofaciens HL1 (HL1), and groups orally administrated with fermented milk product FS or FM (FS and FM groups). Compared with NC group, we found that PC group and FM group performed higher spontaneous alteration rate in the Y-maze task, while the HL1 group demonstrated lower escape latency in Morris water maze task on days 3 and 4. At the molecular level, we observed that both HL1 and FM groups could significantly downregulate IL-1β level in serum, TNF-α level in the liver, TNF-α and MDA levels in the brain, while upregulate SOD activity in the brain compared to the NC group. Moreover, FM group demonstrated significantly increase in butyric acid in cecal content compared to the NC group. Finally, we used qPCR assay to analyze the change of cecal microbiota composition. Compared to NC group, FM group had a lower proportion of Clostridium perfringens.

In summary, a novel fermented milk product fermented with Lb. kefiranofaciens HL1 and Lc. lactis subsp. cremoris APL015 was developed, offering several processing advantages including desirable chewing properties, consistent texture, ease of transport, and storage stability. Furthermore, the fermented milk product displayed notable anti-oxidative, anti-inflammatory, and anti-aging properties.