Next Article in Journal
The Effects of Digital Marketing of Unhealthy Commodities on Young People: A Systematic Review
Previous Article in Journal
Dietary Habits and Eating Practices and Their Association with Overweight and Obesity in Rural and Urban Black South African Adolescents
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Nutrients
Volume 10
Issue 2
10.3390/nu10020146
nutrients-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Bone Response to Dietary Co-Enrichment with Powdered Whole Grape and Probiotics

by
Cynthia Blanton
Nutrition and Dietetic Programs, Idaho State University, 921 S 8th Ave., Pocatello, ID 83209, USA
Nutrients 2018, 10(2), 146; https://doi.org/10.3390/nu10020146
Submission received: 11 November 2017 / Revised: 6 January 2018 / Accepted: 27 January 2018 / Published: 29 January 2018
Browse Figures
Versions Notes

Abstract

:
Nutrition is a primary modifiable determinant of chronic noncommunicable disease, including osteoporosis. An etiology of osteoporosis is the stimulation of bone-resorbing osteoclasts by reactive oxygen species (ROS). Dietary polyphenols and probiotics demonstrate protective effects on bone that are associated with reduced ROS formation and suppressed osteoclast activity. This study tested the effect of dietary enrichment with powdered whole grape and probiotics (composed of equal parts Bifidobacterium bifidum, B. breve, Lactobacillus casei, L. plantarum, and L. bulgaricus) on bone microarchitecture in a mouse model of age-related osteoporosis. Groups (n = 7 each) of 10-month-old male mice were fed one of six diets for 6 months: 10% grape powder with sugar corrected to 20%; 20% grape powder; 1% probiotic with sugar corrected to 20%; 10% grape powder + 1% probiotic with sugar corrected to 20%; 20% grape powder + 1% probiotic; 20% sugar control. Femur, tibia and 4th lumbar vertebrae from 10-month-old mice served as comparator baseline samples. Bone microarchitecture was measured by micro-computed tomography and compared across diet groups using analysis of variance. Aging exerted a significant effect on tibia metaphysis trabecular bone, with baseline 10-month-old mice having significantly higher bone volume/total volume (BV/TV) and trabecular number measurements and lower trabecular spacing measurements than all 16-month-old groups (p < 0.001). Neither grape nor probiotic enrichment significantly improved bone microarchitecture during aging compared to control diet. The combination of 20% grape + 1% probiotic exerted detrimental effects on tibia metaphysis BV/TV compared to 10% grape + 1% probiotic, and trabecular number and trabecular spacing compared to 10% grape + 1% probiotic, 1% probiotic and control groups (p < 0.05). Femur metaphysis trabecular bone displayed less pronounced aging effects than tibia bone, but also showed detrimental effects of the 20% grape + 1% probiotic vs. most other diets for BV/TV, trabecular number, trabecular spacing and trabecular pattern factor (p < 0.05). Tibia and femur diaphysis cortical bone (cortical wall thickness and medullary area) displayed neither aging nor diet effects (p > 0.05). Vertebrae bone showed age-related deterioration in trabecular thickness and trabecular spacing and a trend toward preservation of trabecular thickness by grape and/or probiotic enrichment (p < 0.05). These findings demonstrate no benefit to bone of combined compared to independent supplementation with probiotics or whole grape powder and even suggest an interference of co-ingestion.
Keywords:
grapes; probiotics; bone; mice; aging

1. Introduction

Bone protection is paramount as people are living longer and must maintain optimal skeletal health into advanced age. A 2015 analysis of US National Health and Nutrition Examination Survey data reports the prevalence of low bone mass as 48% and osteoporosis as 16% in adults over age 65 years [1]. Despite this high rate, osteoporosis is an underdiagnosed and undertreated disease, often not recognized until bone fracture occurs [2,3,4]. Strikingly, up to 50% of vertebral fractures incidentally discovered by radiography occur in patients without prior diagnosis of osteoporosis [5]. By 2025, an expected three million osteoporosis-related fractures will cost the US health care system $25 billion [6]. Prevention is key to reducing the personal and societal burdens of osteoporosis.
Nutrition intervention offers an inexpensive, readily accessible, preventive strategy against osteoporosis that is most effective when adopted early in life [7]. Observational population studies showing bone-protective effects of fruit consumption [8,9,10,11,12,13,14,15,16,17,18] have been supported by mechanistic animal and cellular investigations demonstrating an inhibitory effect of fruit polyphenols on osteoclast formation and activity [19,20,21,22,23,24]. For example, in a cross-sectional study of 3089 Chinese men and women of age 40–75 years old, Qui et al. [8] found significantly higher total and site-specific bone mass density (BMD) in participants consuming the highest vs. lowest tertile of fruit. This effect was consistent for femoral neck BMD across five of six subcategories of fruit, including the grapes/litchi/longan subcategory. From the perspective of fracture risk, Byberg et al. [10] examined the effect of fruit and vegetable intake over a mean 14.2 years in a cohort of 75,591 Swedish men and women. This group reported a 1.39 hazard ratio for hip fracture in people consuming zero vs. 3 servings of fruit per day. Welch et al. [17] focused on the effect of dietary flavonoids, a group of plant compounds with biologic activity, on bone density in 3160 women from the TwinsUK cohort. Results showed a positive association between total flavonoid intake and higher spine BMD. Further, the strongest beneficial effect on hip and spine BMD was seen for high intake of anthocyanins, a subclasss of flavonoids that are concentrated in grapes.
Grapes are one of the most concentrated sources of dietary polyphenols [25] and they have attracted considerable attention as a nutrition intervention against degenerative disease, including osteoporosis. Grape intake is thought to protect bone by blocking inflammatory molecules [26] that stimulate osteoclasts (bone-degrading cells) [20,21,22] and by promoting activity of osteoblasts (bone-building cells) [23,24,27,28]. This mechanism is consistent with the concept of age-related bone loss being largely due to chronic low-grade inflammation [29,30,31]. Grape polyphenol isolates, such as resveratrol, have been widely used to investigate their effect on bone [18]. Resveratrol dietary supplementation demonstrates bone-protective effects in multiple experimental models including obese men at high risk of bone loss [32], mice exposed to oxidative stress [33], ovariectomized rats (a model of post-menopausal osteoporosis) [34], rats with periodontal bone disease [35], and tail-suspended rats [36]. Although whole grape powder is more representative of a typical diet than polyphenol extracts, a grape-enriched diet has been tested in only one known study of bone health. Hohman et al. [37] showed significantly greater femur thickness and strength in ovariectomized rats fed a 25% grape vs. control diet for eight weeks. Further study of the bone response to whole grape and grape powder consumption is needed to inform dietary recommendations specific to bone preservation.
A novel strategy to enhance the bone-protective effects of grape consumption is to increase the production of bioactive grape polyphenol metabolites in the gastrointestinal tract. Intestinal bacteria (microbiota) metabolize dietary polyphenols to compounds that are absorbed into the body and exert beneficial biologic effects [38,39,40]. Bacterial species that metabolize polyphenols to bioactive compounds have been identified and among these are species used as dietary probiotics. Probiotics are health-promoting bacteria added to foods such as yogurt that colonize the intestine [41] and transform undigested food components into biologically active compounds [40,41,42]. Research demonstrates that the most commonly consumed probiotic bacteria produce bioactive secondary metabolites from polyphenol-rich fruit [43,44,45,46], and mouse models indicate that these secondary metabolites of polyphenols augment the health benefits of polyphenol-rich food consumption, producing a synergistic effect [42,47,48]. The purpose of this study was to test the hypothesis that adding probiotics to a grape-enriched diet would augment the bone-protective effects of grape consumption.

2. Methods

2.1. Experimental Design

The experiment was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health. The use of animals and the study protocol were approved by the Idaho State University Animal Care and Use Committee on 21 July 2016 under protocol number 743. Male 8-month-old ND4 Swiss Webster retired breeder mice were purchased from Envigo, Indianapolis, IN and were housed individually in one environmentally controlled room under a 12-h light-dark cycle and fed standard chow for two months. At age 10 months, a group (n = 6) of mice was used to provide bones for baseline reference measurements to which age- and diet-related changes in 16-month-old mice were compared. The remaining groups (n = 7) were fed one of six diets for 6 months (to age 16 months): 10% grape powder with sugar corrected to 20%; 20% grape powder; 1% probiotic with sugar corrected to 20%; 10% grape powder + 1% probiotic with sugar corrected to 20%; 20% grape powder + 1% probiotic; and 20% sugar control.

2.2. Diets

Isonitrogenous diets were created based on a commercially available rodent chow (AIN-93M) modified with freeze-dried grape powder made from whole grapes and probiotics, (Table 1). Grape powder was provided by the California Table Grape Commission. The 20% grape powder concentration is similar to that used in three rodent studies of bone response using grape [37] or plum [49,50] powder. The 20% grape dose equates to a fresh grape intake of 4 servings per day in a 60-kg human [51]. The 10% grape powder diet was included to test for bone effects from a smaller dose.
The probiotic was a mixture of equal parts Bifidobacterium bifidum, B. breve, Lactobacillus casei, L. plantarum, and L. bulgaricus (Nutraceutix®, Redmond, OR, USA) at a concentration of 1011 cfu/g. These strains were selected because they demonstrate growth and polyphenol metabolite production when cultured in polyphenol medium [44,45,46,52,53]. The 1% probiotic concentration provided 1.0 × 108 cfu/g diet, or 5 × 108 cfu/day for a mouse consuming 5 g diet/day, a dose effective in producing bone improvements in mice in our laboratory’s previous study [54]. Fresh batches of diet were prepared monthly at the same time, in the sequence of control, grape, grape + probiotic, and probiotic to minimize the risk of probiotic transfer to non-probiotic diets. Diets were stored frozen and replenished in food dishes daily to ensure viability of probiotics consumed. Our laboratory has previously demonstrated good stability/viability of probiotics mixed into powdered diet [55,56].
Mouse body weight and food intake were measured weekly. At the conclusion of the feeding experiment, mice were euthanized using carbon dioxide and femur, tibia and 4th lumbar spine vertebra were excised. Bones were cleaned of muscle, fixed in 10% formalin for 48 h, and stored in 70% ethanol at 4 °C until micro-computed tomography scanning.

2.3. Micro-Computed Tomography (µCT) Scanning of Bones

High-resolution µCT scans were performed at the University of Utah Small Animal Imaging Core Facility. Bones were scanned on a Siemens Inveon tri-modality (CT/PET/SPECT) system (Siemens AG, Munich, Germany) by a trained technician dedicated to the project. The energy settings used were 150 uA/80 kEv, with an exposure time of 2300 ms. The scans used a full rotation step-and-shoot acquisition with 600 total projections. The reconstructions from the acquired projections resulted in images with an isotropic spatial resolution of 20.49 µm with a matrix size of 1024 × 1024 × 3968.
One operator performed all of the analysis. Reconstructed images were loaded into Siemens Inveon Research Workstation or IRW (Siemens AG, Munich, Germany) software and checked for artifacts, such as motion artifacts and beam hardening effects. Once the reconstructed image quality was deemed acceptable, the desired bone regions (femur, tibia and vertebrae) were cropped and analyzed separately as follows. For the femur, the medial notch superior to the condyles was used as a mean to rotate the femur such that the condyles aligned with the horizontal axis. The tibia was rotated such that the flat posterior side was aligned with the horizontal axis whereas the vertebrae was aligned such that its anterior side was aligned with the horizontal axis. For cortical bone analyses, the femur and tibia were analyzed at the 50% mid-shaft whereas the vertebra was analyzed at the 50% mid-vertebrae region. For the data to reflect the area, a range of 21 slices (±10 slices from the 50% slice) was segmented. Trabecular bone analyses for the femur were performed in the distal femoral metaphysis region defined at 0.1 mm proximal to the distal femoral growth plate extending 1 mm towards the mid diaphysis. Tibia trabecular analyses were performed in the proximal tibia metaphysis defined at 0.1 mm distal to the growth plate of the proximal tibia extending 1 mm towards the diaphysis. Two additional regions were analyzed for the vertebrae, namely the regions at the 25% and 75%, for these additional ranges of interest (ROIs), a range of 15 slices was chosen however the bone morphometric (including the trabecular measurements) results were averaged to accurately reflect the entire vertebra. Measurement for each of the three ROIs was based on the total length of the vertebra measured from the cranial end following orientation. For the purposes of segmentation, a thresholding value based on the image volume histogram that selected 65% of the brightest of pixels was used. This value clearly differentiated the cortical bone from the trabecular bone and bone marrow and was kept consistent from specimen to specimen.
Once each region was segmented, the Siemens IRW software (version 4.2, Munich, Germany) calculated bone morphometric measurements which included the ratio of total trabecular bone volume to total tissue (bone + marrow) volume (BV/TV, in %); the ratio of trabecular bone surface area to trabecular total bone volume (BS/TV, in µm−1), representing the proportion of bone surface available for remodeling; trabecular thickness (Tb.Th, in µm); trabecular number (Tb.N, in µm−1), in which a higher number suggests stronger bone structure; trabecular spacing (Tb.Sp, in µm−1), in which a smaller number indicates better structure; and trabecular pattern (Tb.Pf, in µm−1), which is a measure of connectiveness of trabecular structures and a smaller number indicates more connected bone. Cortical wall thickness for each of the bones was measured manually using a transparency placed over the images, centered on the medullary area of each slice from the 50% region. Eight measurements were collected from the medial, lateral, anterior and posterior planes and the average was calculated to provide one measurement. Whole-bone (whole-femur, -tibia and -vertebrae) measurements of each parameter were calculated using the average of the regional measurements.
Femur, tibia and spine vertebrae were chosen for analysis because they display significant defects in microarchitecture in humans [1] and mice [49,50,54] experiencing age- and estrogen-deficiency-related bone loss and because they showed significant positive effects of probiotic and polyphenol-rich fruit feeding in previous studies conducted by our laboratory [54,56] and other investigators [37,49,50,57,58].

2.4. Data Analysis

Statistical power analysis determined that a sample size of six mice per group provided a >80% probability of detecting a significant difference in femur trabecular bone volume/total volume area (a primary outcome measure of the reduction in bone mass in aging mice) [49,59] between the diet groups.
Data were examined for normal distribution and equality of variances using the Shapiro–Wilk test and analysis of means for variances with Levene test, respectively. Data that were not normally distributed were transformed to log normal prior to analyses. One-way analysis of variance (ANOVA) followed by Tukey–Kramer HSD post-hoc multiple comparisons were used to compare group means when variances were equal. Nonparametric one-way ChiSquare test followed by Wilcoxon nonparametric comparisons for each pair were used when variances were not equal. Descriptive data are reported as means with standard deviations. Percent body weight gain was calculated as: (final (age 16 mo) body weight—initial (age 10 mo) body weight)/initial body weight × 100. Feed efficiency was calculated as (body weight in g/(food intake in g × kcals per g)), using kcals/g for each diet as indicated in Table 1. Final body weight and % body weight gain did not have statistically significant effects on bone outcomes when included as random variables in ANOVAs and thus neither was included in the model. JMP Pro 13 statistical software (Cary, NC, USA) was used to perform data analysis.

3. Results

3.1. Body Weight and Food Intake

Initial body weight, final body weight and % change in body weight were not significantly different across diet groups (p = 0.99, 0.51, and 0.66, respectively), Table 2. However, mixed models ANOVA with repeated measures showed a significant effect of diet group on body weight across weeks (p < 0.0001), with 10% grape + 1% probiotic and 10% and 20% grape groups weighing more than 1% probiotic (p < 0.0001, p = 0.0002 and p = 0.0065, respectively), and 10% grape and 10% grape + 1% probiotic weighing more than control (p = 0.0197 and p = 0.0042, respectively), Figure 1.
Food intake was significantly different across diet groups when analyzed by one-way ANOVA of average intake during the entire experiment and ANOVA with repeated measures across the 24 weeks of the experiment (p < 0.0001 for both), Table 2 and Figure 2. Mice in the 1% probiotic consumed significantly more food per day than mice in the 10% grape, 20% grape, and 20% grape + probiotic groups (p = 0.0003, p < 0.0001 and p = 0.0002, respectively). Although mice in the 1% probiotic group ate the most food per day, feed efficiency was significantly less in the 1% probiotic vs. 10% grape, 10% grape + probiotic and 20% grape + probiotic groups (p < 0.0001 for all post-hoc comparisons).
Mixed models ANOVA with repeated measures showed a significant main effect of diet group on body weight (BW in g) across weeks (p < 0.0001), with 10% grape + 1% probiotic and 10% and 20% grape groups weighing more than 1% probiotic (p < 0.0001, p = 0.0002 and p = 0.0065, respectively), and 10% grape and 10% grape + 1% probiotic weighing more than control (p = 0.0197 and p = 0.0042, respectively).

3.2. Bone Microarchitecture

3.2.1. Tibia

Aging exerted a significant effect on tibia metaphysis trabecular bone, with baseline 10-month-old mice having significantly higher BV/TV and trabecular number measurements and lower trabecular spacing measurements than all 16-month-old groups (p < 0.001) (Table 3). Neither grape nor probiotic enrichment improved bone microarchitecture compared to control diet. The combination of 20% grape + 1% probiotic exerted detrimental effects on tibia metaphysis BV/TV compared to 10% grape + 1% probiotic, and trabecular number and trabecular spacing compared to 10% grape + 1% probiotic, 1% probiotic and control groups (p < 0.05). Tibia cortical bone showed no significant effects of aging or diet (p > 0.05).

3.2.2. Femur

Femur trabecular bone did not demonstrate significant aging effects when comparing 10-month-old mice to 16-month-old mice fed control diet (p > 0.05) (Table 4). Neither grape nor probiotic enrichment improved bone microarchitecture compared to control diet. The combination of 20% grape + 1% probiotic tended to produce detrimental effects on femur metaphysis compared to the other diet groups in that BV/TV, trabecular number, trabecular spacing and trabecular pattern factor for 20% grape + 1% probiotic, but not for most of the other diet groups, were significantly different from 10-month-old mice (p < 0.05). Femur cortical bone showed no significant effects of aging or diet (p > 0.05).

3.2.3. Vertebrae

Vertebrae trabecular bone showed limited effects of aging and diet. The baseline 10-month-old mice displayed the smallest vertebrae trabecular spacing (group main effect p = 0.0394) and the largest trabecular thickness (group main effect, p = 0.0247; post-hoc analysis baseline vs. control, p < 0.05).

4. Discussion

The hypothesis tested in this study, that probiotics would enhance bone-protective outcomes of grape-enriched diets, was not supported by these data. Results show that combining probiotics with 10% and 20% grape diets exerted either a null or an interfering effect on bone microarchitecture measures in comparison to independent probiotic and grape dietary enrichment. The underlying rationale for the hypothesis was based on evidence showing that polyphenol-rich diets protect bone from age- and post-menopausal-related loss [37,49,50,57], and that select lactobacillus and bifidobacteria strains metabolize polyphenols to bioactive metabolites [38,39,40,60,61,62,63,64,65]. Further, prior studies in animals and humans demonstrate that secondary metabolites of polyphenols produced by gut bacteria, including lactobacillus and bifidobacteria strains, augment the health benefits of polyphenol-rich food consumption [42,43,66,67,68]. Evidence supports that grape consumption protects bone by inhibiting production of inflammatory molecules that stimulate osteoclasts [20,21,22] and by promoting activity of osteoblasts [23,24,27,28]. This mechanism aligns with the concept of age-related bone loss being related to chronic low-grade inflammation and oxidative stress [29,30,31,69,70,71,72]. Zhang et al. [69] showed that biomarkers of oxidative damage to proteins and lipids were significantly higher in plasma and femur from older (18-month-old) vs. young (2-month-old) and adult (6-month-old) rats, and that this age-related increase in oxidative stress was correlated with significantly lower femur bone mineral density (BMD). A negative correlation between lipid peroxidation and femur BMD has also been demonstrated in post-menopausal women with osteoporosis [73].
Several studies suggest a beneficial synergism between probiotics and polyphenol-rich diets. Duoeonne et al. [74] described an increase in circulating microbial metabolites of cranberry polyphenols when mice were co-supplemented with the probiotic Bacillus subtilis CU1. Hakansson et al. [47] reported improved anti-inflammatory and anti-oxidative biomarker levels in colitic mice fed blueberry husks and rye fiber with lactobacillus and bifidobacteria probiotics compared to without probiotics. Similarly, reductions in tissue and blood inflammatory markers resulting from a 4% powdered green tea leave diet fed to mice were augmented by co-ingestion of L. plantarum [42]. While Jakesevic et al. [43] showed no significant enhancement in anti-oxidative defense following intestinal ischemia-reperfusion in mice when L. plantarum was added to a bilberry-enriched diet, their data do not indicate antagonism of the probiotic on bilberry-associated bioactivity. Interestingly, their study demonstrated that adding L. plantarum to bilberry- and chokeberry-enriched diets prevented reductions in colonic lactobacillus counts caused by the berry diets. The authors suggested that L. plantarum, which possesses enzymatic activity toward polyphenols [75,76], may have altered the colonic microbiota profile to facilitate the growth and maintenance of polyphenol-metabolizing lactobacillus. Modification of the intestinal microbiota by polyphenols has been documented in multiple human and animal studies as well as in vitro models [46,77,78,79,80,81,82]. For example, lactobacillus and bifidobacteria concentrations are increased in response to feeding of red wine polyphenols and flavanol-enriched cocoa powder [83,84].
Reasons for the absence of an observed improvement in bone integrity with the combined probiotic + grape diets in this study may include an interaction between the probiotic strains and commensal gut bacteria, leading to impaired production of polyphenol metabolites that are beneficial to bone. Interactions between polyphenols and other dietary components have been previously reported. Blanton et al. [55] described a partial negation of blood pressure reduction in spontaneously hypertensive rats fed blueberry when probiotics were co-ingested. In that study, blueberry-induced increases in urine hippuric acid levels were not impaired by probiotics, suggesting that polyphenol absorption was not blocked by probiotics. However, hippuric acid is one of many metabolites of polyphenols and the production of other bioactive metabolites could have been affected by the added probiotics [85]. Bolca et al. [86] showed that microbial activation of dietary phytoestrogens into bioactive compounds was modified by co-supplementation with different foods. In addition, an interaction between supplemental vitamin C and grape-seed polyphenols was reported by Ward et al. [87], wherein the combination treatment reversed improvements in systolic blood pressure caused by the individual supplements, resulting in an increase in systolic blood pressure in hypertensive patients.
Another possibility is that a positive effect of grape oligosaccharides and fiber on bone architecture was inhibited by co-ingestion of probiotics. Higher dietary and specifically fruit fiber intake has been shown to protect against femoral neck bone loss in men [88]. Mechanisms include improved mineral solubility and absorption resulting from growth of gut bacteria that ferment oligosaccharides and fiber to short-chain fatty acids [89,90,91]. The growth of commensal gut bacteria that metabolize grape oligosaccharides might have been antagonized by exogenous probiotic bacteria in the grape + probiotic-fed mice in this study.
A different strategy of using probiotics to metabolize polyphenols to bioactive compounds prior to consumption has also been tested. Lambert et al. [92] demonstrated a significant bone benefit in osteopenic post-menopausal women that consumed probiotic-fermented red clover extract (high in isoflavones) for 12 months. In that study, probiotics were allowed to ferment the extract prior to consumption, whereas the present study was designed to promote grape polyphenol fermentation by probiotics within the mouse intestine. Elsewhere, an equol-rich supplement produced from lactococcus garvieae-fermented soy germ showed superior bioavailability to intact soy isoflavones [93]. Consumption of pre-formed polyphenol metabolites thus may be a more reliable method for exploiting probiotic transformation of polyphenols to bioactive compounds than co-ingestion of polyphenol-rich food and probiotics.
The lack of a significant beneficial effect of the 1% probiotic compared to control diet on bone microarchitecture in this study is not consistent with previous findings and may be related to differences in probiotic strains used. Blanton et al. [56] reported significant protection against femur trabecular loss due to skeletal unloading in adult male rats fed a synbiotic diet consisting of Lactobacillus acidophilus, Lactococcus lactis lactis and fructooligosaccharides. McCabe et al. [94] showed significant increases in femoral trabecular parameters in healthy male mice supplemented with Lactobacillus reuteri for four weeks. These improved bone outcomes were associated with lower intestinal expression of TNF-α, a marker of inflammation. The investigators also demonstrated that L. reuteri supplementation suppressed bone resorption and preserved femur trabecular bone parameters in ovariectomized mice [95]. Using a mouse model of type I diabetes-related bone loss, Zhang et al. [96] showed that four weeks of L. reuteri treatment preserved osteoblast function and protected femur trabecular and cortical parameters against loss. Lastly, Lactobacillus paracasei and a mixture of Lactobacillus strains, including L. plantarum, prevented femur cortical loss and reduced the expression of femur TNF-α in ovariectomized mice [97].
It is important to recognize the differences among animal models of osteoporosis in the site-specificity and rate of bone loss, which might impact treatment effects. Age-related osteoporosis, as modeled in the current study, is characterized by a reduced rate of bone turnover [57,98], whereas ovariectomy is marked by a rapid onset of accelerated bone turnover with an increase in bone resorption and relative deficiency in bone formation [99]. Both models of osteoporosis result in the deterioration of trabecular bone, but differences may exist in cortical bone response. Age-related decreases in cortical bone thickness due to expansion of the medullary region have been reported by some [49,100], but not all [59], investigators. In ovariectomized rodents, cortical bone thickness has been shown to decline [97], increase [101], or remain unchanged [102]. Despite these differences, it is notable that polyphenol-rich fruit consumption has produced beneficial bone responses in both models of osteoporosis [37,49,57,103].
The lower body weight, higher food intake, and lower feed efficiency in the 1% probiotic mice are consistent with prior findings from some rodent studies but contrary to those from others, particularly experiments in commercial production animals. McCabe et al. [94] reported a trend toward lower body weight and significantly less visceral fat in mice supplemented with L. reuteri. Similarly, Karlsson et al. [104] showed significantly less 6-month weight gain in rats supplemented with L. plantarum vs. plain water. Our laboratory previously found no significant effects of synbiotic- or probiotic-containing diets in body weight, food intake, or feed efficiency in male rats [55,56]. A meta-analysis of human and animal studies showed varying effects of different Lactobacillus species on weight change, with L. plantarum, a probiotic used in the current study, promoting weight loss [105]. Probiotic supplementation is commonly used to enhance feed efficiency and body weight gain in commercial animal production [106,107].

5. Conclusions

In conclusion, dietary co-enrichment with grape powder and probiotics does not produce a synergistic beneficial bone response in aging mice. Moreover, the data suggest an interaction between grape and probiotics that exerts negative bone effects compared to independent dietary enrichment with either supplement.

Acknowledgments

This work was supported by a grant from the California Table Grape Commission. Freeze-dried whole grape powder was provided by the California Table Grape Commission.

Conflicts of Interest

The author declares no conflict of interest.

References

  1. Looker, A.C.; Frenk, S.M. Percentage of Adults Aged 65 and over with Osteoporosis or Low Bone Mass at the Femur Neck or Lumbar Spine: United States, 2005–2010; U.S. Centers for Disease Control and Prevention Health E-Stat; National Center for Health Statistics: Hyattsville, MD, USA, 2015.
  2. Balasubramanian, A.; Tosi, L.L.; Lane, J.M.; Dirschl, D.R.; Ho, P.R.; O’Malley, C.D. Declining rates of osteoporosis management following fragility fractures in the U.S., 2000 through 2009. J. Bone Jt. Surg. Am. 2014, 96, e52. [Google Scholar] [CrossRef] [PubMed]
  3. Gehlbach, S.H.; Fournier, M.; Bigelow, C. Recognition of osteoporosis by primary care physicians. Am. J. Public Health 2002, 92, 271–273. [Google Scholar] [CrossRef] [PubMed]
  4. Liu, Z.; Weaver, J.; de Papp, A.; Li, Z.; Martin, J.; Allen, K.; Hui, S.; Imel, E.A. Disparities in osteoporosis treatments. Osteoporos. Int. 2015. [Google Scholar] [CrossRef] [PubMed]
  5. Kroth, P.J.; Murray, M.D.; McDonald, C.J. Undertreatment of osteoporosis in women, based on detection of vertebral compression fractures on chest radiography. Am. J. Geriatr. Pharmacother. 2004, 2, 112–118. [Google Scholar] [CrossRef]
  6. Burge, R.; Dawson-Hughes, B.; Solomon, D.H.; Wong, J.B.; King, A.; Tosteson, A. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005–2025. J. Bone Miner. Res. 2007, 22, 465–475. [Google Scholar] [CrossRef] [PubMed]
  7. Li, J.J.; Huang, Z.W.; Wang, R.Q.; Ma, X.M.; Zhang, Z.Q.; Liu, Z.; Chen, Y.M.; Su, Y.X. Fruit and vegetable intake and bone mass in Chinese adolescents, young and postmenopausal women. Public Health Nutr. 2013, 16, 78–86. [Google Scholar] [CrossRef] [PubMed]
  8. Qiu, R.; Cao, W.T.; Tian, H.Y.; He, J.; Chen, G.D.; Chen, Y.M. Greater Intake of Fruit and Vegetables is Associated with Greater Bone Mineral Density and Lower Osteoporosis Risk in Middle-Aged and Elderly Adults. PLoS ONE 2017, 12, e0168906. [Google Scholar] [CrossRef] [PubMed]
  9. Benetou, V.; Orfanos, P.; Feskanich, D.; Michaelsson, K.; Pettersson-Kymmer, U.; Eriksson, S.; Grodstein, F.; Wolk, A.; Bellavia, A.; Ahmed, L.A.; et al. Fruit and Vegetable Intake and Hip Fracture Incidence in Older Men and Women: The CHANCES Project. J. Bone Miner. Res. 2016, 31, 1743–1752. [Google Scholar] [CrossRef] [PubMed]
  10. Byberg, L.; Bellavia, A.; Orsini, N.; Wolk, A.; Michaelsson, K. Fruit and vegetable intake and risk of hip fracture: A cohort study of Swedish men and women. J. Bone Miner. Res. 2015, 30, 976–984. [Google Scholar] [CrossRef] [PubMed]
  11. Chen, Y.M.; Ho, S.C.; Woo, J.L.F. Greater fruit and vegetable intake is associated with increased bone mass among postmenopausal Chinese women. Br. J. Nutr. 2006, 96, 745–751. [Google Scholar] [PubMed]
  12. Lanham-New, S.A. Fruit and vegetables: The unexpected natural answer to the question of osteoporosis prevention? Am. J. Clin. Nutr. 2006, 83, 1254–1255. [Google Scholar] [PubMed]
  13. Liu, Z.M.; Leung, J.; Wong, S.Y.S.; Wong, C.K.M.; Chan, R.; Woo, J. Greater Fruit Intake was Associated with Better Bone Mineral Status among Chinese Elderly Men and Women: Results of Hong Kong Mr. Os and Ms. Os Studies. J. Am. Med. Dir. Assoc. 2015, 16, 309–315. [Google Scholar] [CrossRef] [PubMed]
  14. Prynne, C.J.; Mishra, G.D.; O’Connell, M.A.; Muniz, G.; Laskey, M.A.; Yan, L.; Prentice, A.; Ginty, F. Fruit and vegetable intakes and bone mineral status: A cross sectional study in 5 age and sex cohorts. Am. J. Clin. Nutr. 2006, 83, 1420–1428. [Google Scholar] [PubMed]
  15. Tucker, K.L.; Chen, H.; Hannan, M.T.; Cupples, L.A.; Wilson, P.W.; Felson, D.; Kiel, D.P. Bone mineral density and dietary patterns in older adults: The Framingham Osteoporosis Study. Am. J. Clin. Nutr. 2002, 76, 245–252. [Google Scholar] [CrossRef] [PubMed]
  16. Tucker, K.L.; Hannan, M.T.; Chen, H.; Cupples, L.A.; Wilson, P.W.; Kiel, D.P. Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women. Am. J. Clin. Nutr. 1999, 69, 727–736. [Google Scholar] [PubMed]
  17. Welch, A.; MacGregor, A.; Jennings, A.; Fairweather-Tait, S.; Spector, T.; Cassidy, A. Habitual flavonoid intakes are positively associated with bone mineral density in women. J. Bone Miner. Res. 2012, 27, 1872–1878. [Google Scholar] [CrossRef] [PubMed]
  18. Shen, C.L.; von Bergen, V.; Chyu, M.C.; Jenkins, M.R.; Mo, H.; Chen, C.H.; Kwun, I.S. Fruits and dietary phytochemicals in bone protection. Nutr. Res. 2012, 32, 897–910. [Google Scholar] [CrossRef] [PubMed]
  19. Kim, T.H.; Jung, J.W.; Ha, B.G.; Hong, J.M.; Park, E.K.; Kim, H.J.; Kim, S.Y. The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss. J. Nutr. Biochem. 2011, 22, 8–15. [Google Scholar] [CrossRef] [PubMed]
  20. Moriwaki, S.; Suzuki, K.; Muramatsu, M.; Nomura, A.; Inoue, F.; Into, T.; Yoshiko, Y.; Niida, S. Delphinidin, one of the major anthocyanidins, prevents bone loss through the inhibition of excessive osteoclastogenesis in osteoporosis model mice. PLoS ONE 2014, 9, e97177. [Google Scholar] [CrossRef] [PubMed]
  21. Wattel, A.; Kamel, S.; Prouillet, C.; Petit, J.P.; Lorget, F.; Offord, E.; Brazier, M. Flavonoid quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NF kappa B and AP-1. J. Cell. Biochem. 2004, 92, 285–295. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, J.; Lazarenko, O.P.; Kang, J.; Blackburn, M.L.; Ronis, M.J.; Badger, T.M.; Chen, J.R. Feeding blueberry diets to young rats dose-dependently inhibits bone resorption through suppression of RANKL in stromal cells. PLoS ONE 2013, 8, e70438. [Google Scholar] [CrossRef] [PubMed]
  23. Chen, J.R.; Lazarenko, O.P.; Wu, X.; Kang, J.; Blackburn, M.L.; Shankar, K.; Badger, T.M.; Ronis, M.J. Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/beta-catenin canonical Wnt signaling. J. Bone Miner. Res. 2010, 25, 2399–2411. [Google Scholar] [CrossRef] [PubMed]
  24. Chen, J.R.; Lazarenko, O.P.; Zhang, J.; Blackburn, M.L.; Ronis, M.J.; Badger, T.M. Diet-derived phenolic acids regulate osteoblast and adipocyte lineage commitment and differentiation in young mice. J. Bone Miner. Res. 2014, 29, 1043–1053. [Google Scholar] [CrossRef] [PubMed]
  25. Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jimenez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727–747. [Google Scholar] [PubMed]
  26. Chu, H.; Tang, Q.; Huang, H.; Hao, W.; Wei, X. Grape-seed proanthocyanidins inhibit the lipopolysaccharide-induced inflammatory mediator expression in RAW264.7 macrophages by suppressing MAPK and NF-kappab signal pathways. Environ. Toxicol. Pharmacol. 2015, 41, 159–166. [Google Scholar] [CrossRef] [PubMed]
  27. Hsu, Y.L.; Liang, H.L.; Hung, C.H.; Kuo, P.L. Syringetin, a flavonoid derivative in grape and wine, induces human osteoblast differentiation through bone morphogenetic protein-2/extracellular signal-regulated kinase 1/2 pathway. Mol. Nutr. Food Res. 2009, 53, 1452–1461. [Google Scholar] [CrossRef] [PubMed]
  28. Zhang, Z.; Zheng, L.; Zhao, Z.; Shi, J.; Wang, X.; Huang, J. Grape seed proanthocyanidins inhibit H2O2-induced osteoblastic MC3T3-E1 cell apoptosis via ameliorating H2O2-induced mitochondrial dysfunction. J. Toxicol. Sci. 2014, 39, 803–813. [Google Scholar] [CrossRef] [PubMed]
  29. Khosla, S. Pathogenesis of age-related bone loss in humans. J. Gerontol. A Biol. Sci. Med. Sci. 2013, 68, 1226–1235. [Google Scholar] [CrossRef] [PubMed]
  30. Chung, H.Y.; Lee, E.K.; Choi, Y.J.; Kim, J.M.; Kim, D.H.; Zou, Y.; Kim, C.H.; Lee, J.; Kim, H.S.; Kim, N.D.; et al. Molecular inflammation as an underlying mechanism of the aging process and age-related diseases. J. Dent. Res. 2011, 90, 830–840. [Google Scholar] [CrossRef] [PubMed]
  31. Braun, T.; Schett, G. Pathways for bone loss in inflammatory disease. Curr. Osteoporos. Rep. 2012, 10, 101–108. [Google Scholar] [CrossRef] [PubMed]
  32. Ornstrup, M.J.; Harslof, T.; Kjaer, T.N.; Langdahl, B.L.; Pedersen, S.B. Resveratrol increases bone mineral density and bone alkaline phosphatase in obese men: A randomized placebo-controlled trial. J. Clin. Endocrinol. Metab. 2014, 99, 4720–4729. [Google Scholar] [CrossRef] [PubMed]
  33. Zhao, L.; Wang, Y.; Wang, Z.; Xu, Z.; Zhang, Q.; Yin, M. Effects of dietary resveratrol on excess-iron-induced bone loss via antioxidative character. J. Nutr. Biochem. 2015, 26, 1174–1182. [Google Scholar] [CrossRef] [PubMed]
  34. Mizutani, K.; Ikeda, K.; Kawai, Y.; Yamori, Y. Resveratrol attenuates ovariectomy-induced hypertension and bone loss in stroke-prone spontaneously hypertensive rats. J. Nutr. Sci. Vitaminol. 2000, 46, 78–83. [Google Scholar] [CrossRef] [PubMed]
  35. Bhattarai, G.; Poudel, S.B.; Kook, S.H.; Lee, J.C. Resveratrol prevents alveolar bone loss in an experimental rat model of periodontitis. Acta Biomater. 2016, 29, 398–408. [Google Scholar] [CrossRef] [PubMed]
  36. Habold, C.; Momken, I.; Ouadi, A.; Bekaert, V.; Brasse, D. Effect of prior treatment with resveratrol on density and structure of rat long bones under tail-suspension. J. Bone Miner. Metab. 2011, 29, 15–22. [Google Scholar] [CrossRef] [PubMed]
  37. Hohman, E.E.; Weaver, C.M. A grape-enriched diet increases bone calcium retention and cortical bone properties in ovariectomized rats. J. Nutr. 2015, 145, 253–259. [Google Scholar] [CrossRef] [PubMed]
  38. Bolca, S.; Van de Wiele, T.; Possemiers, S. Gut metabotypes govern health effects of dietary polyphenols. Curr. Opin. Biotechnol. 2013, 24, 220–225. [Google Scholar] [CrossRef] [PubMed]
  39. Van Duynhoven, J.; Vaughan, E.E.; Jacobs, D.M.; Kemperman, R.A.; van Velzen, E.J.; Gross, G.; Roger, L.C.; Possemiers, S.; Smilde, A.K.; Dore, J.; et al. Metabolic fate of polyphenols in the human superorganism. Proc. Natl. Acad. Sci. USA 2011, 108 (Suppl. 1), 4531–4538. [Google Scholar] [CrossRef]
  40. Williamson, G.; Clifford, M.N. Colonic metabolites of berry polyphenols: The missing link to biological activity? Br. J. Nutr. 2010, 104 (Suppl. 3), S48–S66. [Google Scholar] [CrossRef]
  41. O’Toole, P.W.; Cooney, J.C. Probiotic bacteria influence the composition and function of the intestinal microbiota. Interdiscip. Perspect. Infect. Dis. 2008, 2008, 175285. [Google Scholar] [CrossRef] [PubMed]
  42. Axling, U.; Olsson, C.; Xu, J.; Fernandez, C.; Larsson, S.; Strom, K.; Ahrne, S.; Holm, C.; Molin, G.; Berger, K. Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice. Nutr. Metab. 2012, 9, 105. [Google Scholar] [CrossRef] [PubMed]
  43. Jakesevic, M.; Aaby, K.; Borge, G.I.; Jeppsson, B.; Ahrne, S.; Molin, G. Antioxidative protection of dietary bilberry, chokeberry and Lactobacillus plantarum HEAL19 in mice subjected to intestinal oxidative stress by ischemia-reperfusion. BMC Complement. Altern. Med. 2011, 11, 8. [Google Scholar] [CrossRef] [PubMed]
  44. Molan, A.L.; Lila, M.A.; Mawson, J.; De, S. In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts. World J. Microbiol. Biotechnol. 2009, 25, 1243–1249. [Google Scholar] [CrossRef]
  45. Selma, M.V.; Espin, J.C.; Tomas-Barberan, F.A. Interaction between phenolics and gut microbiota: Role in human health. J. Agric. Food Chem. 2009, 57, 6485–6501. [Google Scholar] [CrossRef] [PubMed]
  46. Vendrame, S.; Guglielmetti, S.; Riso, P.; Arioli, S.; Klimis-Zacas, D.; Porrini, M. Six-week consumption of a wild blueberry powder drink increases bifidobacteria in the human gut. J. Agric. Food Chem. 2011, 59, 12815–12820. [Google Scholar] [CrossRef] [PubMed]
  47. Hakansson, A.; Branning, C.; Adawi, D.; Molin, G.; Nyman, M.; Jeppsson, B.; Ahrne, S. Blueberry husks, rye bran and multi-strain probiotics affect the severity of colitis induced by dextran sulphate sodium. Scand. J. Gastroenterol. 2009, 44, 1213–1225. [Google Scholar] [CrossRef] [PubMed]
  48. Hakansson, A.; Stene, C.; Mihaescu, A.; Molin, G.; Ahrne, S.; Thorlacius, H.; Jeppsson, B. Rose hip and Lactobacillus plantarum DSM 9843 reduce ischemia/reperfusion injury in the mouse colon. Dig. Dis. Sci. 2006, 51, 2094–2101. [Google Scholar] [CrossRef] [PubMed]
  49. Halloran, B.P.; Wronski, T.J.; VonHerzen, D.C.; Chu, V.; Xia, X.; Pingel, J.E.; Williams, A.A.; Smith, B.J. Dietary dried plum increases bone mass in adult and aged male mice. J. Nutr. 2010, 140, 1781–1787. [Google Scholar] [CrossRef] [PubMed]
  50. Rendina, E.; Hembree, K.D.; Davis, M.R.; Marlow, D.; Clarke, S.L.; Halloran, B.P.; Lucas, E.A.; Smith, B.J. Dried plum’s unique capacity to reverse bone loss and alter bone metabolism in postmenopausal osteoporosis model. PLoS ONE 2013, 8, e60569. [Google Scholar] [CrossRef] [PubMed]
  51. Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 2008, 22, 659–661. [Google Scholar] [CrossRef] [PubMed]
  52. Jakesevic, M.; Xu, J.; Aaby, K.; Jeppsson, B.; Ahrne, S.; Molin, G. Effects of bilberry (Vaccinium myrtillus) in combination with lactic acid bacteria on intestinal oxidative stress induced by ischemia-reperfusion in mouse. J. Agric. Food Chem. 2013, 61, 3468–3478. [Google Scholar] [CrossRef] [PubMed]
  53. Peluso, I.; Romanelli, L.; Palmery, M. Interactions between prebiotics, probiotics, polyunsaturated fatty acids and polyphenols: Diet or supplementation for metabolic syndrome prevention? Int. J. Food Sci. Nutr. 2014, 65, 259–267. [Google Scholar] [CrossRef] [PubMed]
  54. Choman, M.; Blanton, C.; Gabaldón, A. The Effect of a Synbiotic Diet on Bone Structure in Aging Male Mice. FASEB J. 2015, 29, 738. [Google Scholar]
  55. Blanton, C.; He, Z.; Gottschall-Pass, K.T.; Sweeney, M.I. Probiotics blunt the anti-hypertensive effect of blueberry feeding in hypertensive rats without altering hippuric acid production. PLoS ONE 2015, 10, e0142036. [Google Scholar] [CrossRef] [PubMed]
  56. Blanton, C.A.; Gabaldon, A.M. Effect of dietary synbiotics on bone in mature male rats following recovery from hindlimb unloading. Int. J. Probiotics Prebiotics 2012, 7, 99–108. [Google Scholar]
  57. Smith, B.J.; Graef, J.L.; Wronski, T.J.; Rendina, E.; Williams, A.A.; Clark, K.A.; Clarke, S.L.; Lucas, E.A.; Halloran, B.P. Effects of dried plum supplementation on bone metabolism in adult C57BL/6 male mice. Calcif. Tissue Int. 2014, 94, 442–453. [Google Scholar] [CrossRef] [PubMed]
  58. Smith, B.J.; Bu, S.Y.; Wang, Y.; Rendina, E.; Lim, Y.F.; Marlow, D.; Clarke, S.L.; Cullen, D.M.; Lucas, E.A. A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat. Bone 2014, 58, 151–159. [Google Scholar] [CrossRef] [PubMed]
  59. Glatt, V.; Canalis, E.; Stadmeyer, L.; Bouxsein, M.L. Age-related changes in trabecular architecture differ in female and male C57BL/6J mice. J. Bone Miner. Res. 2007, 22, 1197–1207. [Google Scholar] [CrossRef] [PubMed]
  60. Sanchez-Patan, F.; Barroso, E.; van de Wiele, T.; Jimenez-Giron, A.; Martin-Alvarez, P.J.; Moreno-Arribas, M.V.; Martinez-Cuesta, M.C.; Pelaez, C.; Requena, T.; Bartolome, B. Comparative in vitro fermentations of cranberry and grape seed polyphenols with colonic microbiota. Food Chem. 2015, 183, 273–282. [Google Scholar] [CrossRef] [PubMed]
  61. Lala, G.; Malik, M.; Zhao, C.; He, J.; Kwon, Y.; Giusti, M.M.; Magnuson, B.A. Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats. Nutr. Cancer 2006, 54, 84–93. [Google Scholar] [CrossRef] [PubMed]
  62. He, J.; Magnuson, B.A.; Lala, G.; Tian, Q.; Schwartz, S.J.; Giusti, M.M. Intact anthocyanins and metabolites in rat urine and plasma after 3 months of anthocyanin supplementation. Nutr. Cancer 2006, 54, 3–12. [Google Scholar] [CrossRef] [PubMed]
  63. Forester, S.C.; Waterhouse, A.L. Identification of Cabernet Sauvignon anthocyanin gut microflora metabolites. J. Agric. Food Chem. 2008, 56, 9299–9304. [Google Scholar] [CrossRef] [PubMed]
  64. Faria, A.; Fernandes, I.; Norberto, S.; Mateus, N.; Calhau, C. Interplay between anthocyanins and gut microbiota. J. Agric. Food Chem. 2014, 62, 6898–6902. [Google Scholar] [CrossRef] [PubMed]
  65. Sanchez-Patan, F.; Cueva, C.; Monagas, M.; Walton, G.E.; Gibson, G.R.; Quintanilla-Lopez, J.E.; Lebron-Aguilar, R.; Martin-Alvarez, P.J.; Moreno-Arribas, M.V.; Bartolome, B. In vitro fermentation of a red wine extract by human gut microbiota: Changes in microbial groups and formation of phenolic metabolites. J. Agric. Food Chem. 2012, 60, 2136–2147. [Google Scholar] [CrossRef] [PubMed]
  66. Lampe, J.W.; Chang, J.L. Interindividual differences in phytochemical metabolism and disposition. Semin. Cancer Biol. 2007, 17, 347–353. [Google Scholar] [CrossRef] [PubMed]
  67. Cardona, F.; Andres-Lacueva, C.; Tulipani, S.; Tinahones, F.J.; Queipo-Ortuno, M.I. Benefits of polyphenols on gut microbiota and implications in human health. J. Nutr. Biochem. 2013, 24, 1415–1422. [Google Scholar] [CrossRef] [PubMed]
  68. Williamson, G.; Manach, C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am. J. Clin. Nutr. 2005, 81, 243S–255S. [Google Scholar] [PubMed]
  69. Zhang, Y.B.; Zhong, Z.M.; Hou, G.; Jiang, H.; Chen, J.T. Involvement of oxidative stress in age-related bone loss. J. Surg. Res. 2011, 169, e37–e42. [Google Scholar] [CrossRef] [PubMed]
  70. Mundy, G.R. Osteoporosis and inflammation. Nutr. Rev. 2007, 65, S147–S151. [Google Scholar] [CrossRef] [PubMed]
  71. Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Yilmaz, Z. Effects of calcitonin, risedronate, and raloxifene on erythrocyte antioxidant enzyme activity, lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Arch. Med. Res. 2007, 38, 196–205. [Google Scholar] [CrossRef] [PubMed]
  72. Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Aydogan, R.; Yilmaz, Z. Role of antioxidant systems, lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Mol. Cell. Biochem. 2007, 295, 45–52. [Google Scholar] [CrossRef] [PubMed]
  73. Sendur, O.F.; Turan, Y.; Tastaban, E.; Serter, M. Antioxidant status in patients with osteoporosis: A controlled study. Jt. Bone Spine 2009, 76, 514–518. [Google Scholar] [CrossRef] [PubMed]
  74. Dudonne, S.; Varin, T.V.; Anhe, F.F.; Dube, P.; Roy, D.; Pilon, G.; Marette, A.; Levy, E. Modulatory effects of a cranberry extract co-supplementation with Bacillus subtilis CU1 probiotic on phenoli compounds bioavailability and gut microbiota composition in high-fat diet-fed mice. Pharmanutrition 2015, 3, 89–100. [Google Scholar] [CrossRef]
  75. Barthelmebs, L.; Divies, C.; Cavin, J.F. Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism. Appl. Environ. Microbiol. 2000, 66, 3368–3375. [Google Scholar] [CrossRef] [PubMed]
  76. Osawa, R.; Kuroiso, K.; Goto, S.; Shimizu, A. Isolation of tannin-degrading lactobacilli from humans and fermented foods. Appl. Environ. Microbiol. 2000, 66, 3093–3097. [Google Scholar] [CrossRef] [PubMed]
  77. Collins, S.M. A role for the gut microbiota in IBS. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 497–505. [Google Scholar] [CrossRef] [PubMed]
  78. Cueva, C.; Sanchez-Patan, F.; Monagas, M.; Walton, G.E.; Gibson, G.R.; Martin-Alvarez, P.J.; Bartolome, B.; Moreno-Arribas, M.V. In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: Changes in microbial groups and phenolic metabolites. FEMS Microbiol. Ecol. 2013, 83, 792–805. [Google Scholar] [CrossRef] [PubMed]
  79. Duenas, M.; Munoz-Gonzalez, I.; Cueva, C.; Jimenez-Giron, A.; Sanchez-Patan, F.; Santos-Buelga, C.; Moreno-Arribas, M.V.; Bartolome, B. A survey of modulation of gut microbiota by dietary polyphenols. Biomed. Res. Int. 2015, 2015, 850902. [Google Scholar]
  80. Hervert-Hernandez, D.; Pintado, C.; Rotger, R.; Goni, I. Stimulatory role of grape pomace polyphenols on Lactobacillus acidophilus growth. Int. J. Food Microbiol. 2009, 136, 119–122. [Google Scholar] [CrossRef] [PubMed]
  81. Tzounis, X.; Rodriguez-Mateos, A.; Vulevic, J.; Gibson, G.R.; Kwik-Uribe, C.; Spencer, J.P. Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study. Am. J. Clin. Nutr. 2011, 93, 62–72. [Google Scholar] [CrossRef] [PubMed]
  82. Viveros, A.; Chamorro, S.; Pizarro, M.; Arija, I.; Centeno, C.; Brenes, A. Effects of dietary polyphenol-rich grape products on intestinal microflora and gut morphology in broiler chicks. Poult. Sci. 2011, 90, 566–578. [Google Scholar] [CrossRef] [PubMed]
  83. Dolara, P.; Luceri, C.; De Filippo, C.; Femia, A.P.; Giovannelli, L.; Caderni, G.; Cecchini, C.; Silvi, S.; Orpianesi, C.; Cresci, A. Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats. Mutat. Res. 2005, 591, 237–246. [Google Scholar] [CrossRef] [PubMed]
  84. Jang, S.; Sun, J.; Chen, P.; Lakshman, S.; Molokin, A.; Harnly, J.M.; Vinyard, B.T.; Urban, J.F., Jr.; Davis, C.D.; Solano-Aguilar, G. Flavanol-Enriched Cocoa Powder Alters the Intestinal Microbiota, Tissue and Fluid Metabolite Profiles, and Intestinal Gene Expression in Pigs. J. Nutr. 2016, 146, 673–680. [Google Scholar] [CrossRef] [PubMed]
  85. Mosele, J.I.; Macia, A.; Motilva, M.J. Metabolic and Microbial Modulation of the Large Intestine Ecosystem by Non-Absorbed Diet Phenolic Compounds: A Review. Molecules 2015, 20, 17429–17468. [Google Scholar] [CrossRef] [PubMed]
  86. Bolca, S.; Wyns, C.; Possemiers, S.; Depypere, H.; De Keukeleire, D.; Bracke, M.; Verstraete, W.; Heyerick, A. Cosupplementation of isoflavones, prenylflavonoids, and lignans alters human exposure to phytoestrogen-derived 17beta-estradiol equivalents. J. Nutr. 2009, 139, 2293–2300. [Google Scholar] [CrossRef] [PubMed]
  87. Ward, N.C.; Hodgson, J.M.; Croft, K.D.; Burke, V.; Beilin, L.J.; Puddey, I.B. The combination of vitamin C and grape-seed polyphenols increases blood pressure: A randomized, double-blind, placebo-controlled trial. J. Hypertens. 2005, 23, 427–434. [Google Scholar] [CrossRef] [PubMed]
  88. Dai, Z.; Zhang, Y.; Lu, N.; Felson, D.; Kiel, D.P.; Sahni, S. Association between dietary fiber intake and bone loss in the Framingham Offspring Study. J. Bone Miner. Res. 2017. [Google Scholar] [CrossRef] [PubMed]
  89. Wang, Y.; Zeng, T.; Wang, S.E.; Wang, W.; Wang, Q.; Yu, H.X. Fructo-oligosaccharides enhance the mineral absorption and counteract the adverse effects of phytic acid in mice. Nutrition 2010, 26, 305–311. [Google Scholar] [CrossRef] [PubMed]
  90. Martin, B.R.; Braun, M.M.; Wigertz, K.; Bryant, R.; Zhao, Y.; Lee, W.; Kempa-Steczko, A.; Weaver, C.M. Fructo-oligosaccharides and calcium absorption and retention in adolescent girls. J. Am. Coll. Nutr. 2010, 29, 382–386. [Google Scholar] [CrossRef] [PubMed]
  91. Abrams, S.A.; Griffin, I.J.; Hawthorne, K.M.; Liang, L.; Gunn, S.K.; Darlington, G.; Ellis, K.J. A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am. J. Clin. Nutr. 2005, 82, 471–476. [Google Scholar] [PubMed]
  92. Lambert, M.N.T.; Thorup, A.C.; Hansen, E.S.S.; Jeppesen, P.B. Combined Red Clover isoflavones and probiotics potently reduce menopausal vasomotor symptoms. PLoS ONE 2017, 12, e0176590. [Google Scholar] [CrossRef] [PubMed]
  93. Setchell, K.D.; Zhao, X.; Shoaf, S.E.; Ragland, K. The pharmacokinetics of S-(-)equol administered as SE5-OH tablets to healthy postmenopausal women. J. Nutr. 2009, 139, 2037–2043. [Google Scholar] [CrossRef] [PubMed]
  94. McCabe, L.R.; Irwin, R.; Schaefer, L.; Britton, R.A. Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice. J. Cell. Physiol. 2013, 228, 1793–1798. [Google Scholar] [CrossRef] [PubMed]
  95. Britton, R.A.; Irwin, R.; Quach, D.; Schaefer, L.; Zhang, J.; Lee, T.; Parameswaran, N.; McCabe, L.R. Probiotic L. reuteri treatment prevents bone loss in a menopausal ovariectomized mouse model. J. Cell. Physiol. 2014, 229, 1822–1830. [Google Scholar]
  96. Zhang, J.; Motyl, K.J.; Irwin, R.; MacDougald, O.A.; Britton, R.A.; McCabe, L.R. Loss of Bone and Wnt10b Expression in Male Type 1 Diabetic Mice is Blocked by the Probiotic Lactobacillus reuteri. Endocrinology 2015, 156, 3169–3182. [Google Scholar] [CrossRef] [PubMed]
  97. Ohlsson, C.; Engdahl, C.; Fak, F.; Andersson, A.; Windahl, S.H.; Farman, H.H.; Moverare-Skrtic, S.; Islander, U.; Sjogren, K. Probiotics protect mice from ovariectomy-induced cortical bone loss. PLoS ONE 2014, 9, e92368. [Google Scholar] [CrossRef] [PubMed]
  98. Kiel, D.P.; Rosen, C.J.; Dempster, D. Age-Related Bone Loss. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism; Rosen, C.J., Ed.; American Society of Bone and Mineral Research: Washington, DC, USA, 2008; Volume 98–101. [Google Scholar]
  99. Jee, W.S.; Yao, W. Overview: Animal models of osteopenia and osteoporosis. J. Musculoskelet. Neuronal Interact. 2001, 1, 193–207. [Google Scholar] [PubMed]
  100. Halloran, B.P.; Ferguson, V.L.; Simske, S.J.; Burghardt, A.; Venton, L.L.; Majumdar, S. Changes in bone structure and mass with advancing age in the male C57BL/6J mouse. J. Bone Miner. Res. 2002, 17, 1044–1050. [Google Scholar] [CrossRef] [PubMed]
  101. Shah, F.A.; Stoica, A.; Cardemil, C.; Palmquist, A. Multiscale characterization of cortical bone composition, microstructure, and nanomechanical properties in experimentally induced osteoporosis. J. Biomed. Mater. Res. A 2017. [Google Scholar] [CrossRef] [PubMed]
  102. Alexander, J.M.; Bab, I.; Fish, S.; Muller, R.; Uchiyama, T.; Gronowicz, G.; Nahounou, M.; Zhao, Q.; White, D.W.; Chorev, M.; et al. Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice. J. Bone Miner. Res. 2001, 16, 1665–1673. [Google Scholar] [CrossRef] [PubMed]
  103. Hooshmand, S.; Kern, M.; Metti, D.; Shamloufard, P.; Chai, S.C.; Johnson, S.A.; Payton, M.E.; Arjmandi, B.H. The effect of two doses of dried plum on bone density and bone biomarkers in osteopenic postmenopausal women: A randomized, controlled trial. Osteoporos. Int. 2016, 27, 2271–2279. [Google Scholar] [CrossRef] [PubMed]
  104. Karlsson, C.L.; Molin, G.; Fak, F.; Johansson Hagslatt, M.L.; Jakesevic, M.; Hakansson, A.; Jeppsson, B.; Westrom, B.; Ahrne, S. Effects on weight gain and gut microbiota in rats given bacterial supplements and a high-energy-dense diet from fetal life through to 6 months of age. Br. J. Nutr. 2011, 106, 887–895. [Google Scholar] [CrossRef] [PubMed]
  105. Million, M.; Angelakis, E.; Paul, M.; Armougom, F.; Leibovici, L.; Raoult, D. Comparative meta-analysis of the effect of Lactobacillus species on weight gain in humans and animals. Microb. Pathog. 2012, 53, 100–108. [Google Scholar] [CrossRef] [PubMed]
  106. Liao, S.F.; Nyachoti, M. Using probiotics to improve swine health and nutrient utilization. Anim. Nutr. 2017, 3, 331–343. [Google Scholar] [CrossRef]
  107. Yirga, H. The use of probiotics in animal nutrition. J. Prob. Health 2015, 3, 132. [Google Scholar] [CrossRef]
Nutrients 10 00146 g001 550
Figure 1. Body weight across weeks by diet group. BW=body weight in g; 1% Pro = 1% probiotic diet; 10% G 1% Pro = 10% grape + 1% probiotic diet; 20% G 1% Pro = 20% grape + 1% probiotic diet.
Figure 1. Body weight across weeks by diet group. BW=body weight in g; 1% Pro = 1% probiotic diet; 10% G 1% Pro = 10% grape + 1% probiotic diet; 20% G 1% Pro = 20% grape + 1% probiotic diet.
Nutrients 10 00146 g001
Nutrients 10 00146 g002 550
Figure 2. Food intake across weeks by diet group. FI = food intake in g. 1% Pro = 1% probiotic diet; 10% G 1% Pro = 10% grape + 1% probiotic diet; 20% G 1% Pro = 20% grape + 1% probiotic diet. Mixed models ANOVA with repeated measures showed a significant main effect of diet group on food intake (FI, in g) across weeks (p < 0.0001). Mice in the 1% probiotic consumed significantly more food per day than mice in the 10% grape, 20% grape, and 20% grape + probiotic groups (p = 0.0003, p < 0.0001 and p = 0.0002, respectively).
Figure 2. Food intake across weeks by diet group. FI = food intake in g. 1% Pro = 1% probiotic diet; 10% G 1% Pro = 10% grape + 1% probiotic diet; 20% G 1% Pro = 20% grape + 1% probiotic diet. Mixed models ANOVA with repeated measures showed a significant main effect of diet group on food intake (FI, in g) across weeks (p < 0.0001). Mice in the 1% probiotic consumed significantly more food per day than mice in the 10% grape, 20% grape, and 20% grape + probiotic groups (p = 0.0003, p < 0.0001 and p = 0.0002, respectively).
Nutrients 10 00146 g002
Table
Table 1. Composition of diets.
Table 1. Composition of diets.
Ingredient (g)10% Grape20% Grape10% Grape + 1% Probiotic20% Grape + 1% Probiotic1% ProbioticControl
Casein140.0140.0140.0140.0140.0140.0
Cornstarch520.7520.7510.7510.7510.7520.7
Dextrose50.00.050.00.0100.0100.0
Fructose50.00.050.00.0100.0100.0
Cellulose50.050.050.050.050.050.0
Grape Powder100.0200.0100.0200.00.00.0
Probiotic0.00.010.010.010.00.0
Soybean Oil40.040.040.040.040.040.0
Mineral Salt Mix35.035.035.035.035.035.0
Vitamin Mix10.010.010.010.010.010.0
l-Cystine1.81.81.81.81.81.8
Choline Bitartrate2.52.52.52.52.52.5
Total g1000.01000.01000.01000.01000.01000.0
Total kcals3762.83722.83722.83682.83762.83802.8
Kcals/g3.763.723.723.683.763.80
Kcal values: Starch, sugars = 4 kcals/g; grape powder = 3.6 kcals/g; casein = 4 kcals/g; oil = 9 kcals/g. Grape component was freeze-dried grape powder made from whole grapes. Probiotic was a freeze-dried powder composed of equal parts Bifidobacterium bifidum, B. breve, Lactobacillus casei, L. plantarum, and L. bulgaricus at a concentration of 1.0 × 1011 cfu/g.
Table
Table 2. Body weight, food intake and feed efficiency by diet group.
Table 2. Body weight, food intake and feed efficiency by diet group.
10% Grape20% Grape10% Grape + 1% Probiotic20% Grape + 1% Probiotic1% ProbioticControlMain Effect Diet, p-Value (One-Way ANOVA)Main Effect Diet, p-Value (ANOVA Repeated Measures *)
Initial Body Weight (g)37.5538.8238.1038.6538.1637.480.99-
(3.62)(4.36)(3.68)(4.59)(4.75)(3.56)
Final Body Weight (g)42.2042.3843.3042.1639.7841.140.51<0.0001
(1.97)(3.87)(2.06)(3.48)(2.55)(2.82)
Body Weight Gain (% gain of initial) 12.869.5914.399.595.1010.320.66-
(7.11)(8.21)(10.60)(6.92)(10.45)(10.14)
Food Intake (g/day)5.294.975.855.276.195.72<0.0001<0.0001 *
(0.76) a,b(0.44) b(0.63) b,c(0.77) a,b(0.66) c(0.82) b,c
Feed Efficiency (body weight/kcal/day)2.132.221.922.121.721.87<0.0001<0.0001
(0.36) a,b(0.20) a(0.26) b,c(0.33) a,b(0.19) c(0.30) c
Values are means (SD). Initial body weight: body weight at age 10 mo; final body weight: body weight at age 16 mo; body weight gain calculated as: (final (age 16 mo) body weight—initial (age 10 mo) body weight)/initial body weight × 100; feed efficiency calculated as: body weight in g/(food intake in g*kcals per g), using kcals/g for each diet as indicated in Table 1. * ANOVA across repeated weeks. Values not sharing letter superscripts are significantly different (p < 0.05).
Table
Table 3. Tibia microarchitecture in 10-month-old (baseline) mice and 16-month-old mice fed grape- and/or probiotic-enriched or control diet for 6 months.
Table 3. Tibia microarchitecture in 10-month-old (baseline) mice and 16-month-old mice fed grape- and/or probiotic-enriched or control diet for 6 months.
10-Month-Old Baseline10% Grape20% Grape10% Grape + 1% Probiotic20% Grape + 1% Probiotic1% ProbioticControlMain Effect Group, p-Value
Tibia proximal metaphysis
BV/TV, %12.0 (2.1) a5.9 (3.7) b,c6.4 (3.3) b,c6.1 (2.2) b3.67 (1.8) c6.1 (1.8) b,c6.4 (2.7) b,c0.0007
SA/BV, mm −147.047 (3.356)52.343 (9.674)48.691 (8.348)52.942 (3.672)49.445 (6.944)48.969 (5.534)57.136 (6.211)0.2190
Tb. Th, mm0.043 (0.003)0.0392 (0.007)0.042 (0.007)0.038 (0.003)0.041 (0.006)0.041 (0.004)0.035 (0.004)0.2173
Tb. N, mm−12.817 (0.450) a1.399 (0.774) b,c1.482 (0.661) b,c1.608 (0.512) b0.862 (0.314) c1.458 (0.318) b1.776 (0.558) b0.0002
Tb. Sp, mm0.319 (0.050) a1.345 (1.672) b,c0.793 (0.424) b,c0.626 (0.161) b1.260 (0.497) c0.676 (0.187) b0.575 (0.195) b0.0006
Tb. Patt. Fact, mm−19.481 (3.098)17.980 (7.979)16.242 (8.052)16.413 (5.642)18.852 (4.531)14.671 (5.378)14.895 (2.887)0.1611
Tibia mid- diaphysis
Cortical wall thickness, mm0.263 (0.021)0.256 (0.015)0.256 (0.011)0.258 (0.012)0.258 (0.017)0.258 (0.008)0.260 (0.007)0.9735
Medullary area, mm23.585 (0.332)3.918 (0.288)3.790 (0.293)3.630 (0.399)3.724 (0.278)4.165 (0.245)3.919 (0.308)0.0659
Values are means (SD); n = 7 (20% Grape), n = 6 (10% Grape + 1% Probiotic and 20% Grape + 1% Probiotic), n = 5 (10% Grape, 1% Probiotic and Control). BV/TV, bone volume to total volume fraction; SA/BV, bone surface area to bone volume; Tb.Th, trabecular thickness; Tb. N, trabecular number; Tb. Sp, trabecular spacing; Tb. Patt. Fact, trabecular pattern factor. Values not sharing letter superscripts are significantly different (p < 0.05).
Table
Table 4. Femur microarchitecture in 10-month-old (baseline) mice and 16-month-old mice fed grape- and/or probiotic-enriched or control diet for 6 mo.
Table 4. Femur microarchitecture in 10-month-old (baseline) mice and 16-month-old mice fed grape- and/or probiotic-enriched or control diet for 6 mo.
10-Month-Old Baseline10% Grape20% Grape10% Grape + 1% Probiotic20% Grape + 1% Probiotic1% ProbioticControlMain Effect Group, p-Value
Femur distal metaphysis
BV/TV, %11.9 (2.0) a8.4 (3.4) a,b8.5 (3.8) a,b7.6 (3.5) a,b5.6 (1.7) b7.4 (2.6) a,b7.2 (3.7) a,b0.049
SA/BV, mm−144.183 (2.633)40.412 (4.900)43.594 (3.171)43.613 (1.917)45.437 (4.084)43.105 (7.204)45.709 (2.784)0.523
Tb. Th, mm0.045 (0.003)0.050 (0.006)0.046 (0.003)0.046 (0.002)0.044 (0.004)0.048 (0.009)0.044 (0.003)0.473
Tb. N, mm−12.622 (0.412) a1.641 (0.593) a,b1.821 (0.720) a,b1.645 (0.735) a,b1.266 (0.429) b1.572 (0.424) a,b1.605 (0.735) a,b0.018
Tb. Sp, mm0.344 (0.061) a0.678 (0.431) b0.614 (0.342) a,b1.072 (0.864) b0.802 (0.201) b0.628 (0.188) b0.667 (0.299) a,b0.052
Tb. Patt. Fact, mm−112.439 (3.132) a12.981 (3.031) a,b14.433 (3.293) a,b15.562 (1.944) a,b17.863 (1.650) b15.143 (3.135) a,b16.261 (2.360) a,b0.0373
Femur mid- diaphysis
Cortical wall thickness, mm0.254 (0.027)0.264 (0.031)0.264 (0.015)0.252 (0.019)0.252 (0.017)0.279 (0.034)0.258 (0.010)0.4509
Medullary area, mm26.081 (0.514)6.807 (0.534)6.387 (0.583)6.653 (0.319)6.641 (0.429)6.807 (0.551)6.478 (0.097)0.1659
Values are means (SD), n = 7 (20% Grape), n = 6 (10% Grape + 1% Probiotic and 20% Grape + 1% Probiotic), n = 5 (10% Grape and 1% Probiotic), n = 3 (Control). Values not sharing letter superscripts are significantly different (p < 0.05).

Share and Cite

MDPI and ACS Style

Blanton, C. Bone Response to Dietary Co-Enrichment with Powdered Whole Grape and Probiotics. Nutrients 2018, 10, 146. https://doi.org/10.3390/nu10020146

AMA Style

Blanton C. Bone Response to Dietary Co-Enrichment with Powdered Whole Grape and Probiotics. Nutrients. 2018; 10(2):146. https://doi.org/10.3390/nu10020146

Chicago/Turabian Style

Blanton, Cynthia. 2018. "Bone Response to Dietary Co-Enrichment with Powdered Whole Grape and Probiotics" Nutrients 10, no. 2: 146. https://doi.org/10.3390/nu10020146

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop