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Short term supplementation with cranberry extract modulates gut microbiota in human and displays a bifidogenic effect

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• Published: 06 March 2024

Short term supplementation with cranberry extract modulates gut microbiota in human and displays a bifidogenic effect

• Jacob Lessard-Lord, 
• Charlène Roussel, 
• Joseph Lupien-Meilleur, 
• Pamela Généreux, 
• Véronique Richard, 
• Valérie Guay, 
• Denis Roy & 
• Yves Desjardins 

npj Biofilms and Microbiomes volume 10, Article number: 18 (2024) Cite this article

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Abstract

Cranberry is associated with multiple health benefits, which are mostly attributed to its high content of (poly)phenols, particularly flavan-3-ols. However, clinical trials attempting to demonstrate these positive effects have yielded heterogeneous results, partly due to the high inter-individual variability associated with gut microbiota interaction with these molecules. In fact, several studies have demonstrated the ability of these molecules to modulate the gut microbiota in animal and in vitro models, but there is a scarcity of information in human subjects. In addition, it has been recently reported that cranberry also contains high concentrations of oligosaccharides, which could contribute to its bioactivity. Hence, the aim of this study was to fully characterize the (poly)phenolic and oligosaccharidic contents of a commercially available cranberry extract and eval‎uate its capacity to positively modulate the gut microbiota of 28 human subjects. After only four days, the (poly)phenols and oligosaccharides-rich cranberry extract, induced a strong bifidogenic effect, along with an increase in the abundance of several butyrate-producing bacteria, such as Clostridium and Anaerobutyricum. Plasmatic and fecal short-chain fatty acids profiles were also altered by the cranberry extract with a decrease in acetate ratio and an increase in butyrate ratio. Finally, to characterize the inter-individual variability, we stratified the participants according to the alterations observed in the fecal microbiota following supplementation. Interestingly, individuals having a microbiota characterized by the presence of Prevotella benefited from an increase in Faecalibacterium with the cranberry extract supplementation.

초록

크랜베리는 

다양한 건강 혜택과 연관되어 있으며, 

이는 주로 (폴리)페놀 성분, 특히 플라바노-3-올의 높은 함량에 기인한다고 알려져 있습니다.

 그러나 

이러한 긍정적인 효과를 입증하려는 임상 시험들은 

장내 미생물과 이러한 분자 간의 상호작용으로 인한 높은 개인 간 변이성 때문에 

이질적인 결과를 보여왔습니다. 

실제로 여러 연구에서 이러한 분자들이 

동물 및 체외 모델에서 장내 미생물을 조절하는 능력을 보여주었지만, 

인간 대상에 대한 정보는 부족합니다. 

또한 최근 연구에서 

크랜베리가 생물학적 활성에 기여할 수 있는 

올리고사카라이드 고농도를 함유한다는 보고가 있었습니다. 

따라서 본 연구의 목적은 

상업용 크랜베리 추출물의 (폴리)페놀 및 올리고사카라이드 함량을 완전히 특성화하고, 

28명의 인간 대상에서 장내 미생물을 긍정적으로 조절하는 능력을 평가하는 것입니다. 

4일 만에 (폴리)페놀과 올리고사카라이드가 풍부한 크랜베리 추출물은 

강한 비피도제닉 효과를 유발했으며, 

클로스트리디움(Clostridium)과 아네로부티리쿠움(Anaerobutyricum)과 같은 

부티레이트 생성 세균의 풍부함이 증가했습니다. 

크랜베리 추출물은 

혈장 및 분변 단쇄 지방산 프로필을 변화시켜 

아세테이트 비율이 감소하고 

부티레이트 비율이 증가시켰습니다. 

마지막으로 

개인 간 변이를 특성화하기 위해, 

분변 미생물군집 변화에 따라 참가자를 분류했습니다. 

흥미롭게도, 

Prevotella가 풍부한 미생물군집을 가진 개인은 크랜베리 추출물 

보충 후 Faecalibacterium의 증가를 경험했습니다.

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Introduction

Consumption of cranberry (Vaccinium macrocarpon) is associated with multiple health benefits, notably reducing the incidence of urinary tract infections and preventing cardiovascular and neurodegenerative diseases1,2,3,4,5. These positive effects have been primarily attributed to their high concentrations of (poly)phenols4,6,7. Cranberry is rich in various (poly)phenolic compounds, including phenolic acids, anthocyanins and flavonols, with the most preval‎ent being flavan-3-ols7,8. Notably, cranberry is one of the few dietary sources that contains a specific type of oligomeric flavan-3-ols, called A-type proanthocyanidins7,8.

소개

크랜베리(Vaccinium macrocarpon)의 섭취는

다양한 건강 혜택과 연관되어 있으며,

특히 요로 감염 발생률을 감소시키고

심혈관 및 신경퇴행성 질환을 예방하는 효과가 있습니다1,2,3,4,5.

이러한 긍정적인 효과는 주로

크랜베리에 풍부하게 함유된 (폴리)페놀 성분 때문으로 알려져 있습니다4,6,7.

크랜베리는

페놀산, 안토시아닌, 플라보놀 등

다양한 (폴리)페놀 화합물을 풍부하게 함유하고 있으며,

이 중 가장 주요한 성분은 플라반-3-올입니다7,8.

특히

크랜베리는 특정 유형의 올리고머 플라반-3-올인 A형 프로안토시아니딘을 함유한

드문 식이 원천 중 하나입니다7,8.

The positive effects of (poly)phenols on health were believed to stem from the high antioxidant activity in the host. However, research has shown that these molecules are poorly absorbed (< 10%) in the small intestine and a significant portion (> 90%) reaches the colon9,10,11. Hence, it is believed that polyphenols exert their health effects through their action on the gut microbiota in the colon12,13. Indeed, (poly)phenols interact bidirectionally within the gut microbiota. They directly alter its composition by inhibiting the growth of pathogenic bacteria (antimicrobial effect) and stimulating the growth of beneficial ones (prebiotic-like effect). This dual mode of action led to the introduction of the concept of “duplibiotics” by our research group14. Conversely, the gut microbiota can break down (poly)phenols into bioavailable and potentially bioactive metabolites11,12.

Clinical trials investigating the health effects of cranberries have yielded mixed and varied results, most likely due to the large inter-individual variability in the capacity of the gut microbiota to convert cranberry (poly)phenols into bioavailable and potentially bioactive metabolites15,16,17,18. Previous studies have attempted to classify individuals into distinct groups based on their metabolic profiles following the ingestion of cranberry flavan-3-ols. These classifications were based on the differential production of specific metabolites, such as 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, and were not linked to the different gut enterotypes. This approach sought to gain insights into the underlying factors contributing to the inter-individual variability observed in the health effects of cranberry18. However, it was recently demonstrated that the most abundant (poly)phenols family in cranberry, flavan-3-ols oligomers, are not metabolized by the gut microbiota19,20,21. Therefore, since host microorganisms are unable to directly utilize flavan-3-ols oligomers, they do not strictly correspond to the definition of prebiotic according to Gibson et al. 22. However, these molecules exhibit a strong prebiotic-like effect, that is they have the ability to induce beneficial effects through the modulation of the microbiota14. Most notably, cranberry (poly)phenols can enhance the growth and abundance of Akkermansia muciniphila, a mucosal bacterial species associated with numerous health benefits, including potential anti-obesity effect23,24,25,26,27. As the different metabolic profiles alone cannot explain the inter-individual variability linked to the health benefits derived from cranberry consumption, it is imperative to explore other potential sources of variability. One should not neglect the impact of cranberry consumption on the modulation of gut microbiota composition to explain inter-individual variation.

(폴리)페놀의 건강에 대한 긍정적인 효과는

호스트의 높은 항산화 활동에서 비롯된다고 여겨져 왔습니다.

그러나 연구 결과,

이러한 분자들은 소장에서 흡수율이 낮아(< 10%)

대부분(> 90%)이 대장으로 이동합니다9,10,11.

따라서

폴리페놀은 대장의 장내 미생물에 작용하여 건강 효과를 발휘한다고 추정됩니다12,13.

실제로 (폴리)페놀은

장내 미생물군집 내에서 양방향으로 상호작용합니다.

그들은

병원성 세균의 성장 억제(항미생물 효과)를 통해

미생물군집의 구성을 직접적으로 변화시키고,

유익한 세균의 성장을 촉진하는(프리바이오틱스 유사 효과) 이중 작용을 합니다.

이 이중 작용 방식은

우리 연구 그룹에 의해

“듀플리바이오틱스”라는 개념이 제안되었습니다14.

반면,

장내 미생물군집은

(폴리)페놀을 생체 이용 가능하고 잠재적으로

생물학적 활성을 가진 대사물로 분해할 수 있습니다11,12.

크랜베리의 건강 효과를 조사한 임상 시험은

혼합되고 다양한 결과를 보여주었으며,

이는 주로 장내 미생물이 크랜베리 (폴리)페놀을 생체 이용 가능하고

잠재적으로 생물학적 활성을 가진 대사물로 전환하는 능력의

개인 간 변이성 때문일 가능성이 높습니다15,16,17,18.

이전 연구들은

크랜베리 플라반-3-올 섭취 후 대사 프로파일에 따라 개인을 서로 다른 그룹으로 분류하려는 시도를 했습니다.

이러한 분류는

5-(3′,4′-dihydroxyphenyl)-γ-valerolactone과 같은 특정 대사 산물의 차이에 기반했으며,

장 내장형과 연관되지 않았습니다.

이 접근 방식은

크랜베리의 건강 효과에서 관찰된 개인 간 변이성에 기여하는

근본적인 요인을 이해하려는 시도였습니다18.

그러나 최근 연구에서

크랜베리에서 가장 풍부한 (폴리)페놀 계열인

플라반-3-올 올리고머가 장 미생물에 의해 대사되지 않는다는 것이 밝혀졌습니다19,20,21.

따라서

호스트 미생물이 플라반-3-올 올리고머를 직접 이용할 수 없기 때문에,

이는 Gibson 등22의 정의에 따른 프리바이오틱스의 엄격한 정의에 부합하지 않습니다.

그러나 이러한 분자들은

프리바이오틱스와 유사한 효과를 나타내며,

즉 미생물군집의 조절을 통해 유익한 효과를 유도할 수 있습니다14.

특히

크랜베리 (폴리)페놀은

다양한 건강 혜택과 연관된 점막 세균 종인 

Akkermansia muciniphila의 성장과 풍부도를 증가시킬 수 있습니다.

이는 잠재적인 항비만 효과23,24,25,26,27을 포함합니다.

크랜베리 섭취로 인한 건강 혜택과 관련된 개인 간 변이를 설명하기 위해 대사 프로파일 alone으로는 충분하지 않기 때문에,

다른 잠재적 변이 요인을 탐구하는 것이 필수적입니다.

크랜베리 섭취가

장 미생물군 구성 조절에 미치는 영향을 무시해서는 안 됩니다.

Coleman and Ferreira introduced a paradigm shift regarding the constituents responsible for the bioactivity of cranberry28. Their research suggests that complex carbohydrates, such as arabinoxyloglucan and pectic oligosaccharides, present in high concentrations, may play a significant role in mediating the beneficial effects of cranberry. This highlights the importance of considering these specific oligosaccharides as potential contributors to cranberry’s bioactivity and their impact on health outcomes28. In fact, commercially available flavan-3-ols-rich cranberry extracts contain approximately 15% (w/w) of flavan-3-ols, whereas oligosaccharides themselves represent about 20% of the total extract mass28. This suggests that the oligosaccharides present in cranberry extracts may play a significant role in cranberry’s health benefits, through a prebiotic effect28,29.

Thus, the aim of this study was to eval‎uate the effect of a 4-day supplementation with a purified cranberry extract (PrebiocranTM), containing both (poly)phenols and oligosaccharides, on the composition and function of the fecal microbiota in a human clinical trial involving 39 healthy individuals. Fecal microbiota composition was analyzed by 16 S rRNA sequencing and short-chain fatty acids (SCFA) were quantified in both feces and plasma. Moreover, as part of the study, participants were categorized into enterotypes based on the cranberry extract-induced changes in the abundance of bacterial genera within their gut microbiota. This stratification unravelled specific responses depending on the initial gut microbiota composition and clarified the relationship between cranberry extract supplementation and inter-individual variability.

Coleman과 Ferreira는

크랜베리의 생물학적 활성에 기여하는 구성 성분에 대한

패러다임 전환을 제시했습니다28.

그들의 연구는

아라비노옥시글루칸과 펙틴 올리고사카라이드와 같은 복잡한 탄수화물이 높은 농도로 존재하며,

크랜베리의 유익한 효과를 매개하는 데 중요한 역할을 할 수 있음을 시사합니다.

이는 이러한 특정 올리고사카라이드가

크랜베리의 생물학적 활성에 기여하는 잠재적 요인으로 고려되어야 하며,

건강 결과에 미치는 영향도 중요함을 강조합니다28.

실제로 상업용 플라반-3-올이 풍부한 크랜베리 추출물에는

약 15% (w/w)의 플라반-3-올이 함유되어 있으며,

올리고사카라이드 자체는 총 추출물 질량의 약 20%를 차지합니다28.

이는

크랜베리 추출물에 존재하는 올리고사카라이드가 프리바이오틱 효과28,29를 통해

크랜베리의 건강 혜택에 중요한 역할을 할 수 있음을 시사합니다.

따라서

본 연구의 목적은 39명의 건강한 참가자를 대상으로 한

인간 임상 시험에서 (폴리)페놀과 올리고사카라이드를 모두 함유한 정제된 크랜베리 추출물(PrebiocranTM)의

4일 간 보충이 분변 미생물군집의 구성과 기능에 미치는 영향을 평가하는 것입니다.

분변 미생물군집 구성은

16S rRNA 시퀀싱으로 분석되었으며,

분변과 혈장 내 단쇄 지방산(SCFA)은 정량화되었습니다.

또한 연구의 일환으로, 참가자들은 크랜베리 추출물 섭취로 인한 장내 미생물군집 내 세균 속의 풍부도 변화에 따라 엔테로타입으로 분류되었습니다. 이 분류는 초기 장내 미생물군집 구성에 따라 특정 반응을 밝혀냈으며, 크랜베리 추출물 보충과 개인 간 변이성 간의 관계를 명확히 했습니다.

Results

Cranberry extract contains high quantities of oligosaccharides and (poly)phenols

The cranberry extract supplement used in this study provided 109 mg of (poly)phenols and 125 mg of oligosaccharides per day to the participants (Table 1 and Fig. 1a). Flavan-3-ols (82.3 mg/day) accounted for 75% of the total (poly)phenolic content. However, the extract also contained additional (poly)phenols such as flavonols, phenolic acids and anthocyanins (Fig. 1b). A detailed characterization of the (poly)phenols provided by the cranberry extract is presented in Supplementary Table 1. Monosaccharide units forming the cranberry oligosaccharides were determined after acid hydrolysis (to release monosaccharide units from the oligosaccharides) and consisted mainly of glucose (58%), arabinose (24%), xylose (10%), and galactose (4%) (Fig. 1c). Monomeric units of pectic oligosaccharides, namely galacturonic acid, represented less than 1% of the total oligosaccharides content.

Table 1 Characterization of the (poly)phenols and complex carbohydrates in the cranberry extract

Full size table

Fig. 1: Characterization of the cranberry extract.

a Daily dose of (poly)phenols and oligosaccharides provided by the cranberry extract. Proportion of each (poly)phenols class of the total (poly)phenolic content (b) and of each monosaccharide of the total complex carbohydrates content (c).

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Short-term consumption of cranberry extract strongly modulates the fecal microbiota within four days

39 healthy subjects were included in the trial (Table 2); plasma samples were collected from all of them, while 28 provided fecal samples before (V1) and after (V2) the cranberry extract supplementation. Composition of the fecal microbiota was analyzed by 16S rRNA sequencing and is presented in Supplementary Figs. 1 to 4.

Table 2 Age, gender and anthropometric measurements of the participants enrolled in this study

Full size table

Initially, the impact of a four-day supplementation with the cranberry extract on the fecal microbiota of all participants was assessed. The α-diversity was measured by Shannon index and richness by Chao1 index, while β-diversity was calculated by distance-based redundancy analysis (db-RDA) (Fig. 2). The administration of cranberry extract for four days resulted in a significant increase in species richness (p ≤ 0.05) and a decrease in α-diversity (p ≤ 0.01), indicating reduced evenness among bacterial species, as shown in Fig. 2a. Given the substantial variability in bacterial community composition at the species level among participants, the analysis initially focused on examining general trends across participants at the genus level. This approach allowed for a broader understanding of the patterns and associations within bacterial communities that were more consistent across the study population. Consumption of cranberry extract had a significant impact on microbial β-diversity, as shown in Fig. 2b. The first component of the analysis effectively distinguished samples collected before (V1) and after (V2) cranberry supplementation, explaining 18.5% of the total variability in the microbiota (p ≤ 0.001). This finding suggests that cranberry supplementation influenced the overall composition and structure of the gut microbiota. In the V1 samples, the genera Bacteroides and Prevotella_9 were prominent, while in the V2 samples, the genera Bifidobacterium, Fusicatenibacter, and Blautia exhibited stronger associations (Fig. 2b and Supplementary Fig. 5).

Fig. 2: Global effect of the cranberry extract supplementation on the diversity and richness of the fecal microbiota.

a The Shannon and Chao1 indexes were measured before (V1) and after (V2) cranberry extract supplementation. Each sample is represented by a point and a line connects paired samples from the same subjects between V1 and V2. Statistical significance was assessed with paired t-test adjusted for multiple comparisons using Benjamini & Hochberg method. The center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum. b Bacteria genus-based db-RDA discriminating samples collected before (V1) and after (V2) cranberry extract supplementation. Each sample is represented by a point. Statistical significance was assessed with a permutational multivariate analysis of variance (PERMANOVA). Results of the statistical analysis were represented with asterisks (*p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). % of the total variability explained by each axis is reported in axis titles.

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Bifidobacterium is increased with the cranberry extract at the expense of Bacteroides

To further determine whether the abundance of the microbial genera was affected by the cranberry supplementation, a DESeq analysis was performed (Fig. 3a and Supplementary Table 2). Among the most striking results, the abundance of Bacteroides was significantly reduced by the cranberry extract (p ≤ 0.0001, Fig. 3b), while that of Bifidobacterium was significantly increased (p ≤ 0.001, Fig. 3c), as observed with the db-RDA. The treatment led to a decrease of the abundance of certain genera, including Parabacteroides (p ≤ 0.0001), Prevotella_9 (p ≤ 0.05), and Paraprevotella (p ≤ 0.05). On the other hand, the abundance of the genera Terrisporobacter (p ≤ 0.001), Clostridium (p ≤ 0.001), Clostridium sensu stricto 1 (p ≤ 0.05), Anaerobutyricum (p ≤ 0.05), and Dorea (p ≤ 0.05) was increased by the cranberry supplementation (Fig. 3a and Supplementary Fig. 6). To gain further insights into the effects of the cranberry extract on the microbiota, DESeq analysis was conducted focusing on species belonging to the significantly modulated genera (Fig. 3d and Supplementary Table 3). For Bacteroides, five species were specifically decreased by the cranberry supplementation, namely Bacteroides caccae (p ≤ 0.05), Bacteroides thetaiotaomicron (p ≤ 0.001), Bacteroides uniformis (p ≤ 0.01), Bacteroides vulgatus (p ≤ 0.01) and Bacteroides xylanisolvens (p ≤ 0.001) (Fig. 3d and Supplementary Fig. 7a). For Bifidobacterium, two species were increased with the cranberry supplementation, namely Bifidobacterium adolescentis (p ≤ 0.05) and Bifidobacterium longum (p ≤ 0.05), while there was no significant change (p > 0.05) for Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium catenulatum (Fig. 3d and Supplementary Fig. 7b).

Fig. 3: Impact of cranberry extract supplementation on the abundance of bacterial genera and species using DESeq2 analysis.

Volcano plots highlighting significant genera (a) and species (d) that are modulated by the cranberry extract supplementation. Adjusted p-values were obtained with Wald test adjusted for multiple comparisons using the Benjamini & Hochberg method. Dotted line at adjusted p = 0.05 indicates the statistical significance. b, c Boxplots showing the paired microbiota samples for the selected genera Bacteroides and Bifidobacterium. Statistical significance, as determined on the volcano plot, was represented with asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum.

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Quantitative PCR analysis was conducted to validate that cranberry extract supplementation effectively modulated the absolute abundance of Bifidobacterium and Bacteroides in fecal samples. We confirmed that the number of Bifidobacterium was significantly stimulated by the supplementation (p ≤ 0.01, Fig. 4a, d), while Bacteroides was significantly decreased (p ≤ 0.01, Fig. 4b, d). However, the supplementation did not significantly impact the total bacteria concentration in the fecal samples (Fig. 4c, d).

Fig. 4: Validation of the main effects induced by the cranberry extract supplementation by quantitative PCR.

Results are represented as boxplots showing the paired microbiota samples (a–c) and the difference between V2 – V1 (d) of the selected genera Bifidobacterium and Bacteroides, as well as the total microbiota. Statistical significance was assessed with a paired Wilcoxon test adjusted for multiple comparisons using the Benjamini & Hochberg method. Results of the statistical analysis were represented with asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum.

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To investigate the effect of the cranberry extract supplementation on the function of the gut microbiota, SCFA were quantified in feces (Fig. 5a) and plasma (Fig. 5b) samples. Although no significant differences were observed for the ratio of the three major SCFA, due to high inter-individual variability, interesting trends could still be observed. In both matrices, acetate production was globally decreased (p = 0.1 in feces and p = 0.09 in plasma), while butyrate was increased by the cranberry intake (p = 0.1 in feces and p = 0.09 in plasma) (Fig. 5). In addition, the rise of propionate was only observed in plasma samples following the supplementation with cranberry extract (p = 0.09, Fig. 5b).

Fig. 5: Ratio disparity of the three major SCFA after cranberry extract supplementation.

Results in feces (n = 28, a) and in plasma (n = 39, b) are represented as boxplots. The ratio of each major SCFA (acetate, propionate and butyrate) was calculated by dividing its concentration by the sum of the major SCFA and expressed as percentage. The difference between V2 – V1 was visualized to demonstrate the effect of the cranberry extract supplementation on the different ratios. Each subject is represented as a point on the plot. A dotted line was added at the ratio difference V2 – V1 (%) = 0 to indicate no change between V1 and V2. Statistical significance was assessed with paired Wilcoxon test adjusted for multiple comparisons using the Benjamini & Hochberg method. No statistical difference was found between samples collected at V1 and V2. Center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum.

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Faecalibacterium bloomed depending on the initial microbial composition of the participants following cranberry extract supplementation

Furthermore, we explored the microbiota inter-individual variability associated with cranberry supplementation. The participants enrolled in this study were clustered into two enterotypes (Supplementary Fig. 8), based on the principal component analysis (PCA) using the difference in the relative abundance of genera detected in their fecal microbiota (relative abundance of genus X at V2 – relative abundance of genus X at V1). The two enterotypes were obtained following k-means clustering based on the PCA. The first principal component (PC1) separated the two clusters, explaining over 21% of the total variation on the score plot (Supplementary Fig. 8a). As represented on the loading plot (Supplementary Fig. 8b), the partition of the two enterotypes was mainly guided by the modulation of Faecalibacterium and Prevotella_9, and to a lesser extent by other genera, such as Agathobacter (genus comprising the important former Eubacterium rectale), Phocaeicola and Bacteroides. Following the cranberry supplementation, enterotype 1 was characterized by an increase of the abundance of Faecalibacterium (p ≤ 0.05) and Agathobacter (p = 0.3) and a reduction of Prevotella_9 (p ≤ 0.0001), while enterotype 2 was characterized by a greater reduction in the abundance of Phocaeicola (p = .2) and Bacteroides (p = 0.2) (Fig. 6a and Supplementary Fig. 9). Among the 28 participants included in the study, eight individuals were classified as belonging to enterotype 1, while the remaining 20 individuals were assigned to enterotype 2.

Fig. 6: Impact of the cranberry extract supplementation on fecal microbiota depending on the enterotypes.

a Boxplots of the main bacterial genera discriminating the two enterotypes are represented. Statistical significance was assessed with Wilcoxon test adjusted for multiple comparisons using the Benjamini & Hochberg method. Center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum. db-RDA performed with subjects from enterotype 1 (b, c) and enterotype 2 (d, e). Each sample is represented by a point. % of the total variability explained by each axis is reported between parenthesis in axis titles. Statistical significance was assessed with a permutational multivariate analysis of variance (PERMANOVA). Results of the statistical analysis were represented with asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

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However, no significant difference between the two enterotypes (p > .05) was observed for the effect of the cranberry extract on Shannon α-diversity, and Chao1 richness indexes (Supplementary Fig. 10). The effect of the treatment on β-diversity was also assessed by performing a db-RDA on each enterotype separately (Supplementary Fig. 6b–e). This parameter was significantly affected (p ≤ .001) for both enterotypes, with the first component separating samples from V1 and V2 and explaining 43.6% for enterotype 1 and 22.6% for enterotype 2 of the total microbiota variation. For both enterotypes, Bacteroides was associated with V1, while Bifidobacterium and Blautia were linked with V2 (Fig. 6b–e). Remarkably, the bifidogenic effect induced by cranberry consumption was preserved in both enterotypes. However, for enterotype 1, Prevotella_9 was also related to V1 and Faecalibacterium, Dorea and Agathobacter were associated with V2 (Fig. 6b–e).

In order to understand the origin of these differences in the modulation of the fecal microbiota by the cranberry extract supplementation, DESeq analysis was performed with samples collected before the supplementation to assess the initial difference between the enterotypes (Fig. 7a and Supplementary Table 4). Interestingly, Bacteroides was significantly more abundant in subjects belonging to enterotype 2 (Fig. 7b, p ≤ 0.05), while Prevotella_9 was almost exclusively present in samples associated to the enterotype 1 (Fig. 7c, p ≤ 0.001). In fact, only two participants out of twenty within enterotype 2 had a low abundance of this genus (Fig. 7c). Other genera discriminated the initial fecal microbiota (V1) of participants from enterotype 1 from those from enterotype 2 (Supplementary Fig. 11).

Fig. 7: Characterization of the microbial dissimilarity abundance between enterotypes, prior to cranberry extract supplementation using DESeq2 analysis.

a Volcano plot highlighting significant genera (adjusted p > 0.05) that are different between enterotypes prior to the cranberry extract supplementation. Adjusted p-values were obtained with Wald test adjusted for multiple comparisons using the Benjamini & Hochberg method. Dotted line at adjusted p = .05 indicates the statistical significance. b, c Boxplots showing the paired microbiota samples for the selected genera Bacteroides and Prevotella_9. Statistical significance, as determined on the volcano plot, was represented with asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum.

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Finally, we investigated whether there were enterotype-based differences in the production of SCFA in feces (Fig. 8a) and plasma (Fig. 8b). Comparable ratio differences (V2 – V1) for the three major SCFA were observed in fecal samples across both enterotypes, characterized by a consistent decrease in acetate levels and an increase in butyrate concentrations (Fig. 8a). Nonetheless, the cranberry extract appeared to have varying effects on plasmatic SCFA ratios between the two enterotypes, although these differences did not reach statistical significance. (Fig. 8b, p > 0.05). In enterotype 1 individuals, the cranberry extract treatment resulted in a global decrease in acetate levels and an increase in the proportion of propionate. Conversely, within enterotype 2 individuals, the opposite trend was observed, with a slight increase in acetate and a mild decrease in propionate proportions due to the treatment, but high inter-individual variability was observed. In addition, the ratio of butyrate was slightly increased by the cranberry extract supplementation for enterotype 1, while there was no clear modulation for enterotype 2.

Fig. 8: Ratio disparity of the three major SCFA between enterotypes after cranberry extract supplementation.

Results in feces (a) and in plasma (b) are represented as boxplots. The ratio of each major SCFA (acetate, propionate and buryrate) was calculated, as in Fig. 5, by dividing its concentration by the sum of the major SCFA and expressed as percentage. The difference between V2 – V1 was visualized to demonstrate the effect of the cranberry extract supplementation on the different ratios for each enterotype. Each subject is represented as a point on the plot. A dotted line was added at the ratio difference V2 – V1 (%) = 0 to indicate no change between V1 and V2. Statistical significance was assessed with a paired Wilcoxon test adjusted for multiple comparisons using the Benjamini & Hochberg method. No statistical difference was found between enterotypes, nor between samples collected at V1 and V2 for each enterotype. Center of the boxplots represents the median, the borders represent the first and third quartiles and the whiskers represent the minimum and maximum.

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Discussion

Health effects of cranberry have historically been attributed to the antioxidant activity of the (poly)phenols in the host9. Since these molecules are poorly absorbed in the small intestine, the focus has shifted to their interaction with the gut microbiota12,13. It was initially believed that cranberry (poly)phenols, especially flavan-3-ols, could be metabolized by specific gut microbiota members into smaller bioavailable and potentially bioactive metabolites, such as phenyl-γ-valerolactones. However, cranberry flavan-3-ols are mainly A-type oligomeric proanthocyanidins, which are poorly degraded in the gut19,21. Therefore, the purpose of the present study was to fully characterize a commercially available cranberry extract (PrebiocranTM) to determine the nature and the amount of (poly)phenols and oligosaccharides contained in the extract and to assess their capacity to positively modulate the gut microbiota of 28 healthy subjects within a 4-day supplementation.

Cranberry extract contains a complex mixture of (poly)phenols and oligosaccharides, but few studies assessed the oligosaccharides content of cranberry29,30,31,32,33,34,35. Interestingly, oligosaccharides were more abundant than (poly)phenols in the purified cranberry extract used in our study. Monosaccharide composition following acid hydrolysis of oligosaccharides in the cranberry extract is coherent with that reported by Sun et al. in purified oligosaccharides fraction from pectinase-treated cranberry pomace30. Oligosaccharides in this fraction were mostly composed of glucose (47%), arabinose (25%), xylose (23%) and galactose (5%)30, while the oligosaccharides in the cranberry extract used in our study were mainly formed of the same monosaccharides, but with the following proportions: 58%, 24%, 10% and 4%. Hence, we confirmed that the majority of the oligosaccharides in the cranberry extract were arabinoxyloglucans. Only a small amount of galacturonic acid was detected in the cranberry extract (< 1% of the total oligosaccharide content), indicating that pectic oligosaccharides were only present in small amounts. As previously reported for cranberry, flavan-3-ols, particularly A-type proanthocyanidins, were the most abundant (poly)phenol class in the extract7,8. The combined action of (poly)phenols and oligosaccharides likely contributes to the overall impact of cranberry extract on the gut microbiota.

It is worth noting that hypromellose (hydroxypropyl methyl cellulose), the filling agent used in the capsules, could also have an impact on the gut microbiota. Naimi et al. reported that hypromellose decreased bacterial density and α-diversity in an in vitro model36. However, we did not observe that effect in our study.

To our knowledge, this is the first study to demonstrate the effect of a short-term supplementation with cranberry extract, containing both (poly)phenols and oligosaccharides, on the fecal microbiota of human subjects. Interestingly, the consumption of the cranberry extract successfully modulated the fecal microbiota of the participants included in this study with a strong bifidogenic effect. This effect is commonly associated with supplementation of prebiotic fibers, such as inulin and fructooligosaccharides, as first reported by Gibson & Roberfroid37 and confirmed by many other studies38,39,40,41,42. In the present study, Bifidobacterium was significantly increased with the cranberry extract providing low amounts of (poly)phenols (109.3 mg/day) and oligosaccharides (125 mg/day, mainly arabinoxyloglucan). The bifidogenic effect was concomitant to a decrease in Bacteroides abundance, which is recognized to efficiently metabolize complex carbohydrates, such as xylans and arabinoxylans, among others43,44. We surmise that cranberry (poly)phenols have an antimicrobial effect on Bacteroides, allowing Bifidobacterium to consume cranberry oligosaccharides and occupy its microbial niche (prebiotic effect). In fact, the two species of Bifidobacterium that were significantly increased by the cranberry extract, namely B. adolescentis and B. longum, are known to be great degrader of xylo- and arabinoxylan-oligosaccharides45,46. Hence, the combination of (poly)phenols and oligosaccharides in the cranberry extract is coherent with our concept of “duplibiotic”, which is a unabsorbed substrate modulating the gut by both antimicrobial and prebiotic effects14.

Apart from a direct antibacterial effect, the relative reduction of Bacteroides population might result from the symbiotic relationship this genus established with Bifidobacterium in the degradation of cranberry carbohydrates. As it has been demonstrated for arabinogalactan47, this cooperation could lead to an increase in Bifidobacterium population and to the acidification of the medium due to the important concentrations of formate, acetate and lactate produced by this genus when fermenting cranberry xyloglucans48 that could then be detrimental to Bacteroides. Formate, acetate and lactate exert antibacterial effects and pH reduction to which Bacteroides is sensitive49.

Although cranberry extract supplementation resulted in a bifidogenic effect in most subjects, a portion of the subjects did not exhibit the same response to the supplementation. This inherent variability in individuals is a common and frequently observed phenomenon in many supplementation studies. A similar pattern was previously noted in a 4-week study involving daily supplementation of 10 g of oligofructose-enriched inulin50.

The knowledge regarding the effects of cranberry on the gut microbiota in human subjects is relatively limited compared to the abundance of studies conducted in mice and in vitro settings. For instance, a bifidogenic effect was observed following the consumption of cranberry juice (providing 161 mg of (poly)phenols and an unreported amount of oligosaccharides) for 15 days in 10 postmenopausal women51. Along with Bifidobacterium, cranberry juice stimulated the growth of Prevotella, Clostridium XIVa and Eggerthella, while Bacteroides was not affected by the cranberry juice intake. Interestingly, Clostridium XIVa, a known cluster of butyrate-producers, was increased and other studies also reported the stimulation of genera associated with the production of butyrate, such as Eubacterium, Flavonifractor and Subdoligranulum, with cranberry juice supplementation52,53. In our study, Clostridium, Clostridium sensu stricto 1 were also increased, as well another known butyrate-producer, Anaerobutyricum (former Eubacterium) and Shuttleworthia54,55,56,57.

These changes were coherent with the SCFA quantified in both fecal and plasma samples. Indeed, it was observed that the fecal and plasmatic ratio of butyrate tended to increase in response to the cranberry extract, although the differences were not statistically significant. Interestingly, the increase in the butyrate ratio was concomitant to a decrease of the acetate ratio, despite the bloom of several acetate-producing bacteria such as Terrisporobacter (former Clostridium), Intestinibacter (former Clostridium), Dorea (former Eubacterium) and Bifidobacterium58,59,60. However, it has been previously demonstrated that Bifidobacterium promotes the growth and activity of butyrate-producing bacteria, leading to an increase production of butyrate in the gut. Species from Bifidobacterium can consume oligosaccharides to release acetate and lactate, and these intermediate metabolites can be converted into butyrate by specific bacteria, such as Clostridium, by a butyryl-CoA:acetate CoA transferase61,62,63. In the plasma, the increase in the ratio of butyrate was less pronounced compared to that in the feces, whereas the ratio of propionate showed an increase. However, no propionate-producing bacteria was stimulated by the cranberry extract supplementation in this study. Hence, this trend could be explained by the consumption of butyrate by colon epithelial cells, since this substrate is an important energy source for these cells64. Indeed, the decrease in the acetate ratio in the gut, coupled with the utilization of butyrate before it reaches systemic circulation, can indirectly lead to an increase in the ratio of propionate. As a result, the relative proportion of propionate in the bloodstream may appear higher due to these dynamic changes in acetate and butyrate metabolism.

An increase in butyrate production is recognized to be beneficial for human health. In fact, this key metabolite inhibits oxidative stress, inflammation and carcinogenesis in the gut, ameliorates the intestinal barrier and promotes satiety64,65. In addition, butyrate is reported to play a role in the prevention of type 2 diabetes. The microbiota of individuals with type 2 diabetes had a lower abundance of butyrate-producing bacteria; approaches to increase butyrate in the gut are considered promising to regulate glucose in those subjects66. Hence, cranberry extract is a potential prebiotic, as defined by the International Scientific Association for Probiotics and Prebiotics (ISAPP), by stimulating Bifidobacterium, a beneficial bacterial genus, and Bifidobacterium appears to use oligosaccharides from the cranberry extract to favor the production of butyrate, a beneficial metabolite for the gastrointestinal health of the host, by cross-feeding with butyrogenic bacteria22.

In addition to eval‎uating the overall impact of cranberry extract supplementation on the gut microbiota, we conducted enterotype-like clustering based on the changes observed in the fecal microbiota following supplementation. Interestingly, two enterotypes were characterized according to the differential modulation of the microbiota; one corresponded to the Prevotella and the other to the Bacteroides enterotypes previously reported67. In our study, 8 subjects belonged to the Prevotella enterotype (29%) and 20 subjects were included in the Bacteroides enterotype (71%), which is coherent with the reported preval‎ence of Prevotella in Western populations68. The cranberry extract supplementation increased the abundance of Faecalibacterium and Agathobacter in subjects from enterotype 1 (Prevotella enterotype), concomitant to a decrease of Prevotella. In contrast, participants belonging to enterotype 2 (Bacteroides enterotype) only benefited from the global effect, namely the bloom of Bifidobacterium at the expense of Bacteroides. In addition, Phocaeicola and Bacteroides were less reduced by the cranberry extract supplementation for subjects within enterotype 1 than those from enterotype 2. These enterotype-specific changes were coherent with the modulation of the SCFA. In enterotype 1, although not statistically significant, the supplementation led to a more pronounced increase in fecal and plasmatic butyrate ratios and a greater decrease in acetate ratios. This observation may be attributed to the bloom of butyrate-producing genera, such as Faecalibacterium and Agathobacter, within the gut microbiota of individuals belonging to enterotype 169. In fact, it has been demonstrated that these two bacterial genera can produce butyrate by cross-feeding with Bifidobacterium70,71. In addition, the difference in plasmatic ratio of propionate is probably caused by the smaller decrease of propionate-producing bacteria, namely Bacteroides and Phocaiecola (formerly classified as Bacteroides)72,73. Thus, the inter-individual variability associated with the modulation of the fecal microbiota by the cranberry extract, was characterized into enterotypes. These specific enterotypes could explain the variations in the health outcomes resulting from the cranberry extract supplementation, since the gut microbiota of some individuals may produce more butyrate, a beneficial metabolite.

Interestingly, the effect of the initial composition of the gut microbiota on its differential response to oligosaccharides has previously been reported. In fact, the gut microbiota of elderly subjects was found to be modulated differently by arabinoxylan-oligosaccharides from wheat bran, depending on their enterotypes, specifically whether they belonged to the Prevotella or Bacteroides enterotype74. Interestingly, these molecules are able to induce a bifidogenic effect as observed with the cranberry extract75,76. However, in contrast to the cranberry extract, wheat bran oligosaccharides increased the abundance of Prevotella in participants belonging to the Prevotella enterotype74.

Surprisingly, A. muciniphila was not stimulated by the cranberry extract in our study. Previously, our group demonstrated that the abundance of this mucosal bacterial species, associated with antiobesity effect, was increased in mice fed with obesogenic diet supplemented with cranberry extract23,24,25,77. Nonetheless, the present study revealed that the impact on A. muciniphila was limited to individuals who already had this bacterium present in their microbiota prior to the administration of cranberry extract. This subset of samples accounted for approximately 46% of the total samples at V1 (Supplementary Fig. 12a). In most of these subjects, the abundance of this beneficial mucosal bacteria was predominantly increased, but, in certain individuals, it was decreased (Supplementary Fig. 12b). A possible explanation is the short duration of the supplementation in our study. A. muciniphila is a challenging bacterium in term of growth and may require longer supplementation to be stimulated. In fact, in our previous studies, A. muciniphila was stimulated after 8 to 9 weeks of cranberry extract supplementation in mice23,24,77. Also, fecal samples are not the best type of samples to assess the effect of the cranberry extract on A. muciniphila, since this bacteria is found in the mucus layer of the gut and the feces mostly represents the commensal bacteria found in the luminal environment25,78. The best sampling method to probe for this bacteria would be to carry a mucosal biopsy, but this technique is highly invasive, expensive and time-consuming79. A very good alternative is in vitro models, such as the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME®), as we previously demonstrated the effect of ω-3 polyunsaturated fatty acids on A. muciniphila in the mucosal microbial niche of the transverse and descending colon using this dynamic fermentation system80. In addition, the absence of a cranberry extract effect on A. muciniphila, when this bacterium is not initially present, emphasizes the need to develop synbiotic combinations with a probiotic strain (such as A. muciniphila) and a potential prebiotic (like the cranberry extract)81.

In conclusion, this study is the first, to the best of our knowledge, to demonstrate the bifidogenic effect of a short-term supplementation with a cranberry extract rich in both (poly)phenols and oligosaccharides in a short-term human clinical trial. Further research should eval‎uate the long-term effect of this treatment, as well as the impact on health. Although not statistically significant, our study revealed interesting trends in short-chain fatty acid (SCFA) ratios, with an increase in the proportion of butyrate. This observation is particularly important as higher levels of butyrate have been linked to improved gastrointestinal health64,65. Also, it would be interesting to eval‎uate the effect of cranberry (poly)phenols and oligosaccharides separately, to validate the hypothesis of the duplibiotic effect of the cranberry extract and to use inulin as a control to compare the efficiency of the cranberry extract with a recognized prebiotic. Finally, larger cohorts are needed to validate the inter-individual variability associated with the modulation of the fecal microbiota by the cranberry extract, especially the bloom of Faecalibacterium for subjects belonging to the Prevotella enterotype.

논의

크랜베리의 건강 효과는

역사적으로 호스트에 존재하는 (폴리)페놀의 항산화 활동에 기인해 왔습니다9.

이러한 분자들은

소장에서 흡수율이 낮기 때문에,

연구의 초점은 장 미생물군과 상호작용으로 옮겨졌습니다12,13.

초기에는 크랜베리 (폴리)페놀, 특히 플라반-3-올이 특정 장내 미생물군에 의해 더 작은 생체 이용 가능하고 잠재적으로 생물학적 활성을 가진 대사산물(예: 페닐-γ-발레롤락톤)로 대사될 수 있다고 믿어졌습니다. 그러나 크랜베리 플라반-3-올은 주로 장에서 잘 분해되지 않는 A형 올리고머 프로안토시아니딘입니다19,21. 따라서 본 연구의 목적은 상업용 크랜베리 추출물(PrebiocranTM)을 완전히 특성화하여 추출물에 포함된 (폴리)페놀과 올리고사카라이드의 성분과 양을 확인하고, 28명의 건강한 대상자에서 4일간의 보충 후 장내 미생물군집을 긍정적으로 조절하는 능력을 평가하는 것입니다.

크랜베리 추출물은 (폴리)페놀과 올리고사카라이드의 복잡한 혼합물을 함유하지만, 크랜베리의 올리고사카라이드 함량을 평가한 연구는 거의 없습니다29,30,31,32,33,34,35. 흥미롭게도, 본 연구에서 사용된 정제된 크랜베리 추출물에서는 올리고사카라이드가 (폴리)페놀보다 더 풍부했습니다. 크랜베리 추출물의 올리고당을 산 분해 후 단당류 구성은 Sun 등30이 펙틴아제 처리된 크랜베리 찌꺼기에서 정제된 올리고당 분획에서 보고한 것과 일치합니다. 이 분획의 올리고사카라이드는 주로 글루코스(47%), 아라비노스(25%), xylose(23%) 및 갈락토스(5%)로 구성되었습니다30, 반면 본 연구에서 사용된 크랜베리 추출물의 올리고사카라이드는 동일한 단당류로 주로 구성되었지만, 다음과 같은 비율을 보였습니다: 58%, 24%, 10% 및 4%. 따라서, 크랜베리 추출물 내 올리고사카라이드의 대부분이 아라비노크시글루칸임을 확인했습니다. 크랜베리 추출물에서는 갈락투론산이 전체 올리고사카라이드 함량의 1% 미만으로 검출되어, 펙틴 올리고사카라이드가 소량만 존재함을 나타냈습니다. 크랜베리에서 이전에 보고된 바와 같이, 플라반-3-올(특히 A형 프로안토시아니딘)은 추출물에서 가장 풍부한 (폴리)페놀 클래스였습니다7,8. (폴리)페놀과 올리고당류의 복합 작용이 크랜베리 추출물이 장 미생물에 미치는 전체적인 영향에 기여할 가능성이 있습니다.

캡슐의 충진제로 사용된 히프로멜로오스(히드록시프로필 메틸 셀룰로오스)도 장내 미생물에 영향을 미칠 수 있습니다. Naimi 등36은 체외 모델에서 히프로멜로오스가 세균 밀도와 α-다양성을 감소시켰다고 보고했습니다. 그러나 본 연구에서는 해당 효과를 관찰하지 못했습니다.

우리의 지식 범위 내에서, 이 연구는 (폴리)페놀과 올리고사카라이드를 함유한 크랜베리 추출물의 단기 보충이 인간 대상의 분변 미생물군집에 미치는 영향을 입증한 첫 번째 연구입니다. 흥미롭게도, 이 연구에 참여한 참가자들의 분변 미생물군집은 크랜베리 추출물 섭취를 통해 강한 비피도제닉 효과를 통해 성공적으로 조절되었습니다. 이 효과는 인슐린과 프럭토올리고당과 같은 프리바이오틱 섬유 보충과 일반적으로 연관되어 있으며, Gibson & Roberfroid37에 의해 처음 보고되었고 많은 다른 연구38,39,40,41,42에 의해 확인되었습니다. 본 연구에서 크랜베리 추출물은 (폴리)페놀(109.3 mg/일)과 올리고당(125 mg/일, 주로 아라비노옥시글루칸)을 낮은 양으로 함유함에도 불구하고 Bifidobacterium의 수가 유의미하게 증가했습니다. Bifidogenic 효과는 Bacteroides의 풍부도 감소와 동시에 발생했으며, Bacteroides는 xylan과 arabinoxylan과 같은 복잡한 탄수화물을 효율적으로 대사하는 것으로 알려져 있습니다43,44. 우리는 크랜베리 (poly)phenols가 Bacteroides에 대한 항균 효과를 발휘하여 Bifidobacterium이 크랜베리 올리고당을 소비하고 그 미생물 서식지를 차지하도록 허용한다고 추측합니다 (prebiotic 효과). 실제로 크랜베리 추출물에 의해 유의미하게 증가한 두 종의 Bifidobacterium, 즉 B. adolescentis와 B. longum은 xylo- 및 arabinoxylan 올리고당 분해에 뛰어난 미생물로 알려져 있습니다45,46. 따라서 크랜베리 추출물 내 (폴리)페놀과 올리고사카라이드의 조합은 항균 효과와 프리바이오틱 효과를 통해 장을 조절하는 흡수되지 않는 기질이라는 우리 개념인 “듀플리바이오틱”과 일치합니다14.

직접적인 항균 효과 외에도, Bacteroides 군집의 상대적 감소는 이 속이 크랜베리 탄수화물 분해 과정에서 Bifidobacterium과 형성한 공생 관계에서 비롯될 수 있습니다. 아라비노갈락탄47에 대한 연구에서 입증된 것처럼, 이 협력은 이 속이 크랜베리 xyloglucans 발효 시 생성하는 포름산, 아세테이트, 락테이트의 높은 농도로 인해 Bifidobacterium 인구 증가와 매질의 산성화로 이어질 수 있으며, 이는 Bacteroides에 유해할 수 있습니다. 포름산, 아세테이트 및 락테이트는 Bacteroides에 민감한 항균 효과와 pH 감소 효과를 발휘합니다49.

크랜베리 추출물 보충은 대부분의 대상에서 bifidogenic 효과를 나타냈지만, 일부 대상은 보충에 동일한 반응을 보이지 않았습니다. 이 같은 개인 간 변이는 보충 연구에서 흔히 관찰되는 현상입니다. 유사한 패턴은 올리고프루토스 강화 인울린 10g을 매일 보충한 4주 연구에서도 이전에 보고되었습니다50.

인간 대상에서 크랜베리가 장 미생물에 미치는 영향에 대한 지식은 쥐와 체외 실험에서 수행된 연구에 비해 상대적으로 제한적입니다. 예를 들어, 10명의 폐경 후 여성에서 15일 동안 크랜베리 주스(161mg의 (폴리)페놀과 보고되지 않은 양의 올리고사카라이드를 함유)를 섭취한 후 bifidogenic 효과가 관찰되었습니다51. Bifidobacterium과 함께 크랜베리 주스는 Prevotella, Clostridium XIVa 및 Eggerthella의 성장을 자극했으며, Bacteroides는 크랜베리 주스 섭취에 영향을 받지 않았습니다. 흥미롭게도, 부티레이트 생성균으로 알려진 클로스트리디움 XIVa가 증가했으며, 다른 연구에서도 크랜베리 주스 보충이 부티레이트 생성과 관련된 속인 유바크테리움, 플라보니프랙터, 서브돌리그라눌럼의 성장을 자극했다는 보고가 있습니다52,53. 본 연구에서 Clostridium, Clostridium sensu stricto 1도 증가했으며, 다른 알려진 부티레이트 생성균인 Anaerobutyricum (이전 Eubacterium)과 Shuttleworthia도 증가했습니다.54,55,56,57.

이러한 변화는 분변 및 혈장 샘플에서 측정된 SCFA와 일치했습니다. 실제로 크랜베리 추출물에 반응하여 분변과 혈장 내 부티레이트 비율이 증가하는 경향이 관찰되었으나, 차이는 통계적으로 유의미하지 않았습니다. 흥미롭게도, 부티레이트 비율의 증가는 아세테이트 생산 세균인 Terrisporobacter (이전 Clostridium), Intestinibacter (이전 Clostridium), Dorea (이전 Eubacterium) 및 Bifidobacterium58,59,60의 증식에도 불구하고 아세테이트 비율의 감소와 동반되었습니다. 그러나 이전 연구에서 Bifidobacterium이 부티레이트 생성 세균의 성장과 활성을 촉진하여 장 내 부티레이트 생산량을 증가시킨다는 것이 입증되었습니다. Bifidobacterium 속의 종은 올리고사카라이드를 분해하여 아세테이트와 락테이트를 방출하며, 이러한 중간 대사산물은 Clostridium과 같은 특정 세균에 의해 부티릴-코엔자임 A:아세테이트 코엔자임 A 전이효소(butyryl-CoA:acetate CoA transferase)를 통해 부티레이트로 전환됩니다.61,62,63. 혈장에서는 분변에 비해 부티레이트 비율의 증가가 덜 두드러졌으며, 프로피오네이트 비율은 증가했습니다. 그러나 이 연구에서 크랜베리 추출물 보충은 프로피오네이트 생성 세균의 활성화를 유발하지 않았습니다. 따라서 이 경향은 부티레이트가 대장 상피 세포의 중요한 에너지원이기 때문에 이 세포에 의해 부티레이트가 소비되기 때문일 수 있습니다64. 실제로 장 내 아세테이트 비율의 감소와 아세테이트가 체내 순환에 도달하기 전에 부티레이트로 이용되는 현상은 프로피오네이트 비율의 간접적인 증가로 이어질 수 있습니다. 결과적으로 아세테이트와 부티레이트 대사 과정의 동적 변화로 인해 혈중 프로피오네이트의 상대적 비율이 높아질 수 있습니다.

부티레이트 생산량의 증가는 인간 건강에 유익한 것으로 인정됩니다. 실제로 이 핵심 대사산물은 장 내 산화 스트레스, 염증 및 암 발생을 억제하며, 장 장벽을 개선하고 포만감을 촉진합니다64,65. 또한 부티레이트는 제2형 당뇨병 예방에 역할을 한다는 보고가 있습니다. 2형 당뇨병 환자의 미생물군집은 부티레이트 생성 세균의 풍부도가 낮았으며, 장 내 부티레이트를 증가시키는 접근법은 해당 대상의 혈당 조절에 유망한 방법으로 고려되고 있습니다66. 따라서 크랜베리 추출물은 국제 프로바이오틱스 및 프리바이오틱스 과학 협회(ISAPP)가 정의한 대로, 유익한 세균 속인 Bifidobacterium을 자극함으로써 Bifidobacterium은 크랜베리 추출물에서 유래한 올리고사카라이드를 활용해 장내 유익한 대사산물인 부티레이트 생성을 촉진하는 부티레이트 생성 세균과의 교차 영양 작용을 통해 호스트의 소화기 건강에 유익한 부티레이트 생성을 촉진하는 것으로 나타났습니다22.

크랜베리 추출물 보충의 전체적인 장 미생물군집에 대한 영향을 평가하는 것 외에도, 보충 후 분변 미생물군집의 변화에 기반한 엔테로타입 유사 클러스터링을 수행했습니다. 흥미롭게도, 미생물군집의 차이에 따라 두 가지 엔테로타입이 특징지어졌으며, 하나는 Prevotella 엔테로타입에 해당하고 다른 하나는 이전에 보고된 Bacteroides 엔테로타입에 해당했습니다67. 본 연구에서 8명(29%)은 Prevotella 엔테로타입에 속했으며, 20명(71%)은 Bacteroides 엔테로타입에 포함되었습니다. 이는 서양 인구에서 Prevotella의 유병률과 일치합니다68. 크랜베리 추출물 보충은 엔테로타입 1(Prevotella 엔테로타입)에 속한 대상자의 Faecalibacterium과 Agathobacter의 풍부도를 증가시켰으며, 동시에 Prevotella의 감소가 동반되었습니다. 반면, 엔테로타입 2(Bacteroides 엔테로타입)에 속한 참가자는 전체적인 효과만 경험했으며, 이는 Bifidobacterium의 증가와 Bacteroides의 감소로 나타났습니다. 또한, Phocaeicola와 Bacteroides는 enterotype 1에 속한 대상자에서 크랜베리 추출물 보충에 의해 enterotype 2에 속한 대상자보다 덜 감소했습니다. 이러한 enterotype 특이적 변화는 SCFA의 조절과 일치했습니다. enterotype 1에서는 통계적으로 유의미하지는 않았지만, 보충은 분변 및 혈장 내 부티레이트 비율의 더 큰 증가와 아세테이트 비율의 더 큰 감소를 초래했습니다. 이 관찰은 장내 미생물군집에 속하는 개인의 장내 미생물군집에서 부티레이트 생성 속인 Faecalibacterium과 Agathobacter의 증식에 기인할 수 있습니다69. 실제로 이 두 세균 속은 Bifidobacterium과의 교차 영양을 통해 부티레이트를 생성할 수 있다는 것이 입증되었습니다70,71. 또한 혈장 프로피오네이트 비율의 차이는 프로피오네이트 생성 세균인 Bacteroides와 Phocaiecola(이전에는 Bacteroides로 분류됨)의 감소가 더 적기 때문일 가능성이 있습니다.72,73. 따라서 크랜베리 추출물에 의한 분변 미생물군집 조절과 관련된 개인 간 변이는 엔테로타입으로 분류되었습니다. 이러한 특정 엔테로타입은 크랜베리 추출물 보충으로 인한 건강 결과의 변이를 설명할 수 있습니다. 일부 개인의 장 미생물이 유익한 대사산물인 부티레이트를 더 많이 생성할 수 있기 때문입니다.

흥미롭게도 장내 미생물군집의 초기 구성과 올리고사카라이드에 대한 차등 반응 간의 영향은 이전에 보고되었습니다. 실제로 노인 대상자의 장내 미생물군집은 밀겨에서 유래한 아라비노크실란 올리고사카라이드에 의해 엔테로타입에 따라 다르게 조절되었으며, 특히 Prevotella 또는 Bacteroides 엔테로타입에 속하는지에 따라 차이가 있었습니다74. 흥미롭게도 이 분자들은 크랜베리 추출물에서 관찰된 것과 유사한 bifidogenic 효과를 유발할 수 있습니다75,76. 그러나 크랜베리 추출물과 달리 밀겨 올리고사카라이드는 Prevotella 엔테로타입에 속하는 참가자에서 Prevotella의 풍부도를 증가시켰습니다74.

놀랍게도, 우리 연구에서 A. muciniphila는 크랜베리 추출물에 의해 자극되지 않았습니다. 이전 연구에서 우리 연구진은 항비만 효과와 연관된 이 점막 세균 종의 풍부도가 크랜베리 추출물을 보충한 비만 유발 식이를 섭취한 쥐에서 증가했음을 보여주었습니다23,24,25,77. 그럼에도 불구하고, 본 연구에서는 크랜베리 추출물 투여 전 이미 미생물군집에 이 세균이 존재하던 개인에게서만 A. muciniphila에 대한 영향이 제한적임을 밝혔습니다. 이 하위 집합은 V1 시점의 전체 샘플 중 약 46%를 차지했습니다(보조 그림 12a). 이 중 대부분의 대상에서 이 유익한 점막 세균의 풍부도는 주로 증가했지만, 일부 개인에서는 감소했습니다(보조 그림 12b). 이는 본 연구의 보충 기간이 짧았기 때문일 수 있습니다. A. muciniphila는 성장 측면에서 어려운 세균으로, 자극을 위해 더 긴 보충 기간이 필요할 수 있습니다. 실제로 우리 이전 연구에서 A. muciniphila는 쥐에서 크랜베리 추출물 보충 후 8~9주 후에 자극되었습니다23,24,77. 또한 분변 샘플은 크랜베리 추출물이 A. muciniphila에 미치는 영향을 평가하는 데 가장 적합한 샘플 유형이 아닙니다. 이 세균은 장의 점막층에 존재하며, 분변은 주로 장 내강 환경에 존재하는 공생 세균을 대표하기 때문입니다25,78. 이 세균을 조사하기 위한 가장 좋은 채취 방법은 점막 생검이지만, 이 기술은 침습적이며 비용이 많이 들고 시간이 많이 소요됩니다79. 매우 좋은 대안은 체외 모델로, 인간 장 미생물 생태계 점막 시뮬레이터(M-SHIME®)가 있습니다. 우리는 이 동적 발효 시스템을 사용하여 횡행 결장과 하행 결장의 점막 미생물 서식지에서 ω-3 다불포화 지방산이 A. muciniphila에 미치는 영향을 이전에 입증했습니다80. 또한, A. muciniphila가 초기에는 존재하지 않을 때 크랜베리 추출물의 효과가 관찰되지 않은 것은 프로바이오틱 균주(예: A. muciniphila)와 잠재적 프리바이오틱(예: 크랜베리 추출물)을 결합한 시너지틱 조합을 개발할 필요성을 강조합니다81.

결론적으로, 본 연구는 우리 지식 범위 내에서 크랜베리 추출물(폴리페놀과 올리고사카라이드 풍부)의 단기 보충이 단기 인간 임상 시험에서 bifidogenic 효과를 입증한 첫 번째 연구입니다. 추가 연구에서는 이 치료법의 장기적 효과 및 건강에 미치는 영향을 평가해야 합니다. 통계적으로 유의미하지는 않았지만, 본 연구는 단쇄 지방산(SCFA) 비율에서 부티레이트의 비율이 증가하는 흥미로운 경향을 보여주었습니다. 이 관찰은 부티레이트 수치가 높은 것이 위장 건강 개선과 연관되어 있다는 점64,65에서 특히 중요합니다. 또한, 크랜베리 (폴리)페놀과 올리고사카라이드를 각각 분리하여 평가하는 것이 흥미로울 것입니다. 이는 크랜베리 추출물의 듀플리바이오틱 효과를 검증하고, 인슐린을 대조군으로 사용하여 크랜베리 추출물의 효율성을 인정된 프리바이오틱스와 비교하기 위함입니다. 마지막으로, 크랜베리 추출물이 분변 미생물군집을 조절하는 과정에서 나타나는 개인 간 변이성을 검증하기 위해, 특히 Prevotella 엔트로타입에 속하는 대상자에서 Faecalibacterium의 증식 현상을 포함해 더 큰 규모의 코호트 연구가 필요합니다.

Methods

Design of the clinical trial

To assess the impact of a cranberry extract on the fecal microbiota as a proxy for distal luminal colonic microbiota, 39 healthy subjects were enrolled from INAF’s volunteer database, representing the broader community of Quebec City. The selection criteria encompassed various factors, including the maintenance of a stable weight, not-smoking, no pregnancy and/or breastfeeding, following a consistent diet and physical activity routine, refrain from taking medication or experience stability in medication use for at least three months, and abstain from consuming antibiotics and/or probiotics for three months prior to the study. The characteristics of the enrolled subjects are displayed in Table 2.

The participants were instructed to refrain from consuming any food or beverage containing flavan-3-ols, as listed in Supplementary Table 5, for seven days before the intervention. Then, while adhering to these dietary restrictions, they took one cranberry extract capsule (PrebiocranTM) in the morning and one in the evening, providing 109.3 mg of (poly)phenols, as determined by ultra performance liquid chromatography coupled with ultraviolet detector and quadrupole – time of flight (UPLC-UV-QToF), and 125 mg of oligosaccharides per day for 4 days. The daily dose given to patients, comprising two capsules, is equivalent to the extract obtained from 60 g of fresh cranberries, following extraction and purification processes. Prior to and following the supplementation period, samples of feces and plasma were collected with a maximal delay of 2 h between the beginning/end of the treatment and the collection of biological samples. Fecal samples were collected in airtight containers by the participants, which included an anaerobic sachet (Fisher Scientific, Ottawa, Canada) to maintain oxygen-free conditions. The fecal samples were brought to the laboratory after a maximum of 2 h following the donation to prepare a fresh and anaerobic fecal slurry (20%, w/V). Each feces collected were suspended in anaerobic phosphate buffer (8.8 g/L K2HPO4, 6.8 g/L KH2PO4 and 1.0 g/L sodium thioglycolate) and homogenized using a StomacherTM lab blender to remove larger debris as previously described82. Both fecal and plasma samples were aliquoted and promptly stored at −80 °C until subsequent analysis.

This study was approved by the ethics committee for research involving human beings of Laval University under registration number: 2019-312 and informed consent was obtained from all the participants. The study was also registered at https://clinicaltrials.gov/ as NCT05931237.

Characterization of the purified cranberry extract (Prebiocran™)

The purified cranberry extract named Prebiocran™ used in this study was provided by Symrise (Diana Food Canada Inc.). (Poly)phenolic composition was analyzed as previously reported by UPLC-UV-QToF21,83. Monosaccharide composition of oligosaccharides was determined by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) following acid hydrolysis of the cranberry extract. The analysis was done on cranberry extract directly, rather than on the cranberry extract capsules, to avoid interference from the filling agent (hypromellose). Sample preparation was adapted from a previously published methodology30. Briefly, 10 mL of trifluoroacetic acid 2 M in water was added to 10 mg of cranberry extract. The resulting solution was vortexed for 30 s and then incubated at 120 °C for 2 h. Then, the solution was evaporated to dryness under a nitrogen stream, resuspended in 10 mL of isopropanol and re-evaporated to dryness under a nitrogen stream to eliminate residual acid. Before injection, the hydrolyzed cranberry extract was resuspended in 10 mM sodium hydroxide in water to a final concentration of 50 mg/L and passed through a Nylon 0.45 µm filter. The unhydrolyzed cranberry extract was also analyzed to confirm‎ the absence of monosaccharides prior to the acid hydrolysis.

HPAEC-PAD analysis was performed with a DionexTM ICS-6000 (Thermo Scientific, Waltham, MA, USA). 10 µL of sample were injected onto a CarboPac PA20 (3 × 150 mm, 6.5 µm) (Thermo Scientific, Waltham, MA) protected with a guard column CarboPac PA20 (3 × 30 mm, 6.5 µm) (Thermo Scientific, Waltham, MA) heated to 30 °C. Tertiary gradient was performed with water (mobile phase A), 200 mM NaOH in water (mobile phase B) and 200 mM NaOH and 125 mM sodium acetate in water (mobile phase C) at a flow rate of 0.4 mL/min. The gradient started with 97.5% A and 2.5% B, then the proportion of mobile phase B was increased to 100% over 30 minutes. After the elution was done for 15 minutes with 100% C. Finally, the column was washed off for 20 minutes with 100% B and re-equilibrated for 24 minutes with initial conditions. Samples were kept at 8 °C in the autosampler compartment.

DNA extraction

Fecal DNA was isolated from the pellets obtained by centrifuging 500 µL of fecal slurry using the Zymo Research kit (Quick-DNATM Fecal/Soil Microbe MiniPrep Kit) following the manufacturer’s protocol (Zymo Research, Irvine, CA). Enzymatic lysis of DNA was performed using lysozyme (20 mg) and mutanolysine (10 KU) (Sigma-Aldrich, Oakville, Canada). The resulting DNA extracts were eluted in 1X TE buffer (Tris and EDTA) and stored at −20 °C until sequencing. DNA quality was assessed by gel electrophoresis (1.2% w/v agarose) (Life Technologies, Madrid, Spain). DNA concentrations were determined using a Qubit (Thermo Fisher Scientific, Waltham, US). DNA samples were stored at −20 °C until preparation of the 16 S rRNA library.

Fecal microbiota profiling by 16 S rRNA sequencing

The primer pairs F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′) (341F-805R) were employed to amplify the V3-V4 hypervariable region of the 16 S rRNA gene. As suggested by the Qiaseq 16 S Region panel protocol (Qiagen, Hilden, Germany), the QIAseq 16 S/ITS 384-Index I kit (Qiagen) was used to prepare the amplicon library. To assess the quality and size of the 16 S metagenomic libraries, an Agilent High Sensitivity DNA Kit (Agilent, Palo Alto, US) was used in conjunction with a Bioanalyzer. The libraries were quantified using both a Quant-iT PicoGreen dsDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the PCR products were pooled and paired-end sequencing was performed using the MiSeq 600 cycles Reagent Kit V3 on an Illumina MiSeq System (Illumina, San Diego, USA).

The demultiplexed raw data files encompassing all the samples were imported into the R Studio 2022.12.0 environment using R version 4.1.3. The DADA2 R package (version 1.20.0) was employed to infer the amplicon sequence variants (ASV), following the recommended workflow84. Initially, sequence reads underwent filtering and trimming utilizing specific parameters: truncQ = 2, truncLen = c(250, 215) and maxEE = c(2,2). Filtered reads were then subjected to denoising using the DADA2 algorithm, which estimates sequencing errors. Chimeric sequences were removed and the ASV sequences were subsequently merged and classified using the SILVA database SSU Ref NR 99 release 138 with default parameters85. Unassigned taxa and singletons were excluded from the dataset. To account for variations in sampling depth, the data were rescaled to proportions for further analysis.

qPCR assay

The qPCR procedure was conducted on the Applied biosystem 7500 instrument, employing primers targeting 16S total microbiota (Uni334F: 5’-ACTCCTACGGGAGGCAGCAGT-3’; Uni514R: 5’-ATTACCGCGGCTGCTGGC-3’), Bifidobacterium (F: 5’-TCGCGTCYGGTGTGAAAG-3’; R: 5’-CCACATCCAGCRTCCAC-3’) and Bacteroides (F: 5’-GGTGTCGGCTTAAGTGCCAT-3’; R: 5’-CGGAYGTAAGGGCCGTGC-3’)86,87. Each reaction was run in duplicate in a 96-well reaction plate sealed and maintained at a final volume of 15 µL. The qPCR reaction and thermal cycling amplification procedure followed was previously described88. Absolute quantification of gene copy numbers utilized standard curves obtained through droplet digital PCR (ddPCR) on the IBIS (Institute for Integrative Systems Biology, Laval University) sequencing platform, as described previously89.

SCFA analysis in feces and plasma

Feces and plasma were extracted with methyl tert-butyl ether and analyzed by gas chromatography coupled with flame ionization detector (GC-FID) as previously published82,90. Acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and hexanoate were quantified. However, since no significant difference, nor interesting trend were obtained with isobutyrate, valerate, isovalerate and hexanoate, only acetate, propionate and butyrate were reported. SCFA analysis was performed in duplicate. Results were averaged.

Statistical analysis

Statistical analysis was conducted using R version 4.3.0 in the R Studio 2023.03.1 environment. Each measurement was taken from distinct samples. Shannon and Chao1 indexes were calculated with the package vegan (2.6-4). Using the same package, db-RDA was performed with Bray-Curtis distance and the statistical significance of the effect of the cranberry extract supplementation was assessed by permutational multivariate analysis of variance (PERMANOVA). The package DESeq2 (1.40.1) was used to perform differential analysis of normalized counts between conditions (effect of the cranberry extract supplementation or differences between enterotypes) and the Wald test adjusted for multiple comparisons using the Benjamini & Hochberg method was used to assess statistical significance. PCA was carried out using the package FactoMineR (2.8). Clustering was done by k-means algorithm using the package stats (4.3.0). All statistical comparison, except for DESeq analysis, was performed with the package rstatix (0.7.2). Shapiro-Wilk test was performed to assess normality. When data were normally distributed, paired-t test (two-sided) was performed, while Wilcoxon rank sum test (two-sided) was used for non-normal distributions. The paired version of these tests was used to assess the effect of the cranberry extract supplementation (V1 vs V2). Adjustment for multiple comparisons was carried out with Benjamini & Hochberg method. Finally, all graphics were generated with the packages ggplot2 (3.4.2), ggpubr (0.6.0) and ggrepel (0.9.3).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Raw 16S rRNA gene amplicon sequencing data were made publicly available online through the Sequence Read Archive (SRA) portal of NCBI under accession number PRJNA955174.

Code availability

The code used for the bioinformatic and statistical analysis is available upon request to the authors.

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