Humans have been utilizing fermentation as a means of food preservation for thousands of years (Hutkins, 2018). One of the most common substrates for fermentation is milk, with foods such as yogurt and kefir among the oldest fermented foods known (Bourrie et al., 2016; Hutkins, 2018). Kefir in particular is gaining popularity, largely due to an increased appreciation of the potential health benefits. Benefits that have been attributed to specific kefirs include anti-cancer (de Moreno de LeBlanc et al., 2006; Maalouf et al., 2011), immunomodulatory (Vinderola et al., 2005; Lee et al., 2007), cholesterol lowering (Bourrie et al., 2018, 2021), and other metabolic health improvements (Al-Shemmari et al., 2018; Ghizi et al., 2021).

Recently, interest in the health benefits of fermented foods has extended beyond the whole foods to consider the contribution of different fractions and even individual components of the food matrix. Indeed, many food fermentations result in the transformation of food constituents and the production of bioactive components (Marco et al., 2017). Kefir is one of the fermented foods that has had health benefits attributed to multiple different components, which not only includes the fermentation by-products but also a consortium of live microorganisms (yeast and bacteria) that could directly affect the gastrointestinal landscape. For instance, the exopolysaccharides produced by kefir specific microorganisms have been shown to have anti-obesity and immunomodulatory activity (Vinderola et al., 2006a; Lim et al., 2017), while cell-free fractions of kefir have also been shown to have immunomodulatory, lipid lowering, and anti-cancer effects (Vinderola et al., 2006b; Gao et al., 2013; Santanna et al., 2017). Additionally, kefir peptides and peptide fractions have been shown to have a wide range of health impacts including reductions in hyperlipidemia, atherosclerosis, improved neurological function, and reduced non-alcoholic fatty liver disease (NAFLD) risk (Quiros et al., 2005; Chen et al., 2016; Tung et al., 2017, 2020; Malta et al., 2022). There is also some evidence that kefir or kefir components may improve factors associated with atherosclerosis and cardiovascular disease (CVD) development, both of which are associated with obesity and hyperlipidemia (Santanna et al., 2017; Tung et al., 2020; Ghizi et al., 2021).

While traditional kefir is made using kefir grains, pitched kefir is produced by pitching pure cultures microorganisms into milk prior to fermentation. Our group has previously shown that a pitched kefir produced using microorganisms isolated from a traditional kefir could recapitulate the health benefits of traditional beverage in a mouse model of diet-induced obesity (Bourrie et al., 2021). Given that our previous work only examined the impact of whole kefir, we set out to determine whether the microorganisms present in our pitched kefir (PK1), or their components are required for lipid lowering, and if this lipid lowering effect was accompanied by concomitant improvements in CVD and atherosclerosis biomarkers such as vascular cell adhesion molecule 1 (VCAM-1) and C-reactive protein (CRP). To examine this, we fed a high-fat/high-cholesterol diet supplemented with PK1, a centrifuged and filter sterilized cell-free fraction (CFK), and a heat-treated fraction (HK) that underwent a high heat treatment to kill the kefir microbes. We also wanted to determine if freeze-dried kefir consumption would result is health benefits similar to those observed using liquid kefir in our past work. The CFK preparation allowed us to remove all microbes from the kefir and test a fraction that contained no microbes or microbial components, while the HK preparation allowed us to test a fraction in which the microbes had been killed but their microbial components were still present. By comparing how these different kefir preparations impacted weight gain, plasma and liver lipid profiles, short chain fatty acid production, gastrointestinal microbiome composition, and circulating biomarkers for CVD, we hoped to gain insight into how microbial fermentation byproducts may impact the previously described health benefits of our pitched kefir product. Sex differences that may alter the health impacts of PK1were determined by including both male and female mice. We hypothesized that the whole, heat-treated, and filter-sterilized kefir fractions share a common fermentation metabolite that improves plasma and liver lipid profiles, as well as VCAM-1 and CRP.

This is the first study to examine how different kefir preparations consisting of whole kefir, cell-free kefir, and heat-treated kefir impact plasma and liver lipid profiles, the gastrointestinal microbiome, and plasma markers of CVD risk in both males and females using a mouse model of obesity. Given that previous work on traditional kefir had shown that multiple different fractions had positive health impacts (Bourrie et al., 2016), we expected that the CFK and HK treatments in our study would improve lipid and CVD biomarker profiles similarly to PK1 in both male and female mice. Male mice showed a reduction in weight gain across all three kefir treatment groups, while female mice did not have a reduction across any of the treatment groups relative to HFD. Interestingly, both plasma total cholesterol levels were lowered by all kefir preparations in both males and females, while HDL cholesterol was not different among all kefir treatments in females and was only lower in CFK male mice when compared to the HFD control. Non-HDL cholesterol levels were also lower in each kefir treatment in male mice, and in all but the HK group in female mice. This is important as elevated total and non-HDL cholesterol levels are associated with an increased risk of CVD and other metabolic diseases (Després and Lemieux, 2006; Brunner et al., 2019). We also observed that only PK1 and HK reduced VCAM-1 in males. However, this was not seen in the female mice. This may be due to the increased weight gain and generally more severe phenotypes observed in male C57Bl/6 mice when compared to their female counterparts receiving HFD. Indeed, male mice gained approximately 40% more weight than females when fed HFD and VCAM-1 levels in male HFD mice were nearly twice as high as in their female counterparts, while the male LFD group had VCAM-1 levels that were nearly identical to the HFD group in female mice.

In addition to metabolic syndrome and CVD, NAFLD is associated with hyperlipidemia and can lead to steatosis and liver cancer (Loomba and Sanyal, 2013; Duan et al., 2014; Younossi et al., 2018), with increased levels of liver lipids associated with NAFLD risk (Angulo, 2002). We found that liver cholesterol and triglyceride levels were improved by PK1 in both male and female mice. The CFK treatment exhibited a trend for reduction of liver triglycerides in male mice only, and HK did not reduce liver cholesterol or triglycerides in either sex. These results are interesting, as they are contradictory to what we observed in plasma lipid profiles and may indicate that the component of kefir responsible for the observed reduction in liver lipids is somehow removed or reduced during the CFK and HK kefir preparation. One potential reason for these findings may relate to the angiotensin-converting enzyme (ACE) inhibitory action of kefir, which has been attributed to peptides produced from casein during fermentation (Quiros et al., 2005). ACE inhibitory drugs have been found to improve circulating cholesterol and lipid profiles in both human and animal studies (Ravid et al., 1995; Vasiljević et al., 1999; Dost et al., 2014). Some evidence suggests that these drugs may prevent fibrosis and cancer in existing NAFLD; however, more studies are needed, and no studies have examined if ACE inhibitors can prevent the development of NAFLD (Jonsson et al., 2001; Moreno et al., 2010; Li et al., 2018). It is therefore possible that the component of kefir responsible for lowering plasma cholesterol levels is some sort of heat stable peptide with ACE inhibitory activity, while there is a separate component responsible for the improvements in liver lipid levels that is lost or reduced during the preparation of CFK and HK.

Elevated plasma cholesterol, particularly elevated non-HDL cholesterol, has been associated with an increased risk of developing both CVD and diabetes (Scherer and Hill, 2016; Francula-Zaninovic and Nola, 2018; Brunner et al., 2019). One common underlying factor in CVD is the development of atherosclerosis and the associated plaque formation, which can increase an individual’s risk of an adverse cardiovascular event (Francula-Zaninovic and Nola, 2018). Cell adhesion molecules, such as VCAM-1, are commonly used as biomarkers of endothelial function and have been used as indicators of increased CVD and atherosclerosis risk (Springer, 1994; Meng et al., 2021) and play an important role in plaque development (Cybulsky et al., 2001; Ley and Huo, 2001; Varona et al., 2019; Troncoso et al., 2021). We observed a reduction in plasma VCAM-1 in male mice fed both PK1 and HK, but not in the CFK while female mice showed no differences in VCAM-1 between any of the HFD fed groups. The sex difference is likely a result of the less severe atherosclerotic phenotype in female C57BL/6 mice (Paigen et al., 1987), with females in our trial having roughly half the level of VCAM-1 as males. A previous study found that VCAM-1 and plaque formation in the aorta were reduced by a peptide enriched spray dried kefir in HFD fed ApoE^−/− mice (Tung et al., 2020). It is possible that the phenotypes observed in this study would have been more pronounced had we used a more severe phenotype, such as the ApoE^−/− mouse. The lack of effect from CFK treatment may indicate that the compound responsible for this benefit is lost during the centrifugation and filtration process used to create cell-free kefir. Systemic inflammation is another important factor contributing to CVD risk (Vandanmagsar et al., 2011; Boulangé et al., 2016), with CRP being regarded as an important indicator of CVD event risk (Backes et al., 2004; Bisoendial et al., 2010; Avan et al., 2018). Our study found that CRP was not reduced by any of the kefir preparations in either male or female mice fed HFD. This may indicate that, while kefir is able to impact endothelial function positively, the active components associated with these benefits do not impact chronic systemic inflammation as it relates to CRP levels. It is also important to note that, in mice, CRP is only a modest acute phase protein and may not respond to inflammation to the same degree as in humans (Torzewski et al., 2014; Huang et al., 2019). This may be why there has been evidence in human trials that kefir consumption can improve plasma CRP levels despite no corresponding evidence from mouse trials (Ghizi et al., 2021).

All three kefir preparations supplemented to a high-fat and high-cholesterol diet minimally affected the cecal microbiota compared to the high-fat diet alone. PK1 consumption did not increase alpha diversity, as has been observed in a recent study exploring fermented food consumption in a human trial (Wastyk et al., 2021). Absence of an effect on alpha diversity in the current study could be explained by the background diet (HFD vs. uncontrolled) and the controlled environment used for mouse experiments. Due to the lack of fermentable carbohydrates, a high-fat diet can significantly alter gut microbial structure and decrease SCFA concentrations compared to a standard low-fat diet (Dalby et al., 2017). A change in SCFA levels is well documented to correlate with gastrointestinal health and microbiota (Makki et al., 2018; Cong et al., 2022). In agreement, we found higher SCFAs concentrations in the cecum of LFD group with normal cardiovascular health profiles along with changes to the microbial community compared to HFD group. These differences may be due to fermentable fiber content of the low fat and high fat diets, as the low fat diet is made from whole food ingredients containing a variety of fibers while the high fat diet is semi-purified with cellulose as the only fiber. The fact that all three kefir fractions were unable to alter SCFA concentrations or the microbiota suggests that the beneficial effects on cholesterol levels in mice fed kefir is likely not related to SCFA production in the gut. The slight difference in the low abundance microbes in the cecum of female mice but not male mice, as determined by unweighted UniFrac analysis, is likely a result of sex differences in weight, food consumption, locomotor activity, energy expenditure and β cell adaptation upon dietary manipulation (Casimiro et al., 2021). It is also important to note that, while our results did not show significant changes to the cecal microbiome associated with kefir consumption, the composition of the gastrointestinal microbiome of C57BL/6 mice has been shown to differ throughout the gastrointestinal tract (Lkhagva et al., 2021). It is therefore possible that kefir consumption impacts microbiome composition in other sections of the gastrointestinal tract that may contribute to the physiological impacts observed in this study.

This study expands on previous work showing that a pitched kefir (PK1) containing traditional kefir microbes can improve plasma and liver lipid profiles in a mouse model of obesity and that different components of kefir can have beneficial impacts on host health (Maeda et al., 2005; Uchida et al., 2010; Bourrie et al., 2021), however this is the first study to examine different fractions of a pitched kefir in an attempt to differentiate the active components related to lipid control and atherosclerosis improvements. This is also the first study to our knowledge to examine these types of health benefits in both male and female mice, which is of particular importance given the sex differences that exist in both mice and humans with respect to the metabolic responses to obesity and the development of associated metabolic diseases (Paigen et al., 1987; Matyšková et al., 2008; Kautzky-Willer and Handisurya, 2009; Hwang et al., 2010; Sugiyama and Agellon, 2012; Yang et al., 2014). We found that while weight gain was lowered by PK1 consumption in male mice only, each of the kefir treatments lowered plasma total cholesterol and non-HDL cholesterol when compared to a HFD control in both sexes; however, only PK1 treatment resulted in a reduction in liver triglycerides and cholesterol levels in both male and females. These results are interesting as they show that consumption of either cell-free or heat-treated kefir results in similar improvements to plasma cholesterol profiles when compared to PK1 consumption but that improvements in liver lipids requires whole PK1 kefir. This may indicate that live kefir microorganisms are necessary to lower liver lipid content. Additionally, the impact of the kefir treatments for plasma and liver lipids was consistent across males and females, indicating that the mechanism of action is not sex dependent. VCAM-1 was only lowered in male mice receiving PK1 or heat-treated kefir, indicating a possible sex dependent response for this particular biomarker. It is also important to note that female mice had nearly half the levels of VCAM-1 when compared to male mice, and it is possible that with a more severe obesity phenotype there would have been a comparable decrease in plasma VCAM-1 between males and females. Additionally, the lack of improvement in VCAM-1 levels in CFK mice may indicate that, while live microbes are not necessary to lower VCAM-1 in plasma, their microbial components are. These results provide confirmation that reconstituted PK1 kefir containing specific kefir microbes can improve lipid profiles as well as circulating markers of atherosclerosis, and that the lipid lowering benefits are present in both males and females. This is encouraging, as the potential for functional foods that equally benefit male and female individuals is especially important given the increase in females developing metabolic diseases and the need for more research examining these conditions in female populations (Engin, 2017). Future work should focus on identifying how these fractions differ in the composition of their fermentation metabolites and, if possible, on the identification of an active component or components that are responsible for the health benefits observed in these animal trials.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA885841.

The animal study was reviewed and approved by Animal Care and Use Committee at the University of Alberta (AUP 00000671).

BCTB, PDC, CR, and BPW designed research. BCTB, AJF, and TJ conducted research. BCTB and AJF analyzed data. BCTB, AJF, and BPW wrote the paper. BPW had primary responsibility for final content. All authors have read and approved the final manuscript.

This study was directly supported by a grant from the Weston Family Microbiome Initiative. BPW and CR are supported by the Canada Research Chairs Program. Outside of this work, CR also reports grants from the Canadian Institute of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC) and several other agencies. Cotter laboratory is funded by Science Foundation Ireland (SFI) under grant number SFI/12/RC/2273 (APC Microbiome Ireland), by SFI together with the Irish Department of Agriculture, Food and the Marine under grant number SFI/16/RC/3835 (VistaMilk), and by the European Commission under the Horizon 2020 program grant number 818368 (MASTER). Research in the Willing lab is supported by a Natural Sciences and Engineering Discovery Grant (RGPIN-2019-06336).

BCTB, BPW, and PDC hold a patent for the method used to produce the pitched kefir used in the trial.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.