Review on kefir beverages from milk and water: health benefits, processing and applications

Abstract

Kefir is a trending and highly valued fermented beverage known for its beneficial microbes and giving a wide spectrum of health benefits. It includes milk kefir and water kefir which have gained separate attention. Although milk kefir is more heard of than water kefir, it is essential to understand both in greater details in terms of similarities and differences. The key health benefits such as probiotics, protection in the gastrointestinal tract, control of glucose level, antioxidant, antimutagenic, anticarcinogenic, regulating cholesterol, antimicrobial and improving lactose digestion are discussed and supported with recent in-vivo or in-vitro scientific evidences. This review addresses the gap of individual focus of each kefir by direct comparison of its microbial diversity, processing and physicochemical qualities. It provides updated information on lactic acid bacteria by species from advanced techniques of metagenomic analysis and also highlights the potential of kefiran, exopolysaccharides of milk kefir grains in food, biopharmaceutical and packaging industries.

Introduction

Kefir is a fermented drink that has endured a millennia-old history from the Elbrus region, located between southern Russia and northern of the Georgian border expanded to Europe and spread to popularity across the world through commercial routes among people of different cultures who passed on kefir from one generation to another for decades (Ganatsios et al., 2021; Gentry et al., 2023). Kefir is produced through a fermentation process, which undergoes biochemical changes induced by a community of desirable microorganisms at room temperature for approximately one to three days (Bensmira et al., 2019; Hecer et al., 2019; Plessas et al., 2016; Simova et al., 2002). Upon fermentation, kefirs produce a variety of fermentation by-products which include lactic acid, acetic acid, acetaldehyde, ethanol, acetoin, diacetyl, carbon dioxide and several micronutrients, contributing to unique taste and aroma properties (Guzel-Seydim et al., 2000; Lynch et al., 2021; Xu et al., 2019). Kefir can be categorised as milk or water kefir. The milk kefir is more heard of than the water kefir. Although both kefirs have similarities in their symbiotic systems, it is essential to know the unique symbiotic systems that lead to distinct physicochemical qualities (Guzel-Seydim et al., 2021). Figure 1 provides a comparison of the similarities and differences of these two kefirs. Water kefir is prepared using substrate mainly sucrose-containing water with fruits while milk kefir is made using dairy milk sourced from cow, goat or sheep (Rosa et al., 2017; Tzavaras et al., 2022). Upon fermentation, those fermentation compounds contributed to fizzy and fruity taste in water kefir, which is distinct from milk kefir which consists of milky, slightly creamy and tangy flavour (Bueno et al., 2021, Rosa et al.,2017). The kefir grains of both kefirs used as the starter culture differ in terms of appearance, chemical structure and microbial composition. Milk kefir grains are generally described as tiny, gelatinous, translucent, pale yellowish, water-soluble substances, with small clumps of irregular shape and appearance like cauliflower with a range of diameters between 0.3 and 3.5 cm (Garrote et al., 2010; Gaware et al., 2011). Milk kefir grains typically comprise around 50% Lactobacillus spp., 20% Leuconostoc spp., 10% Streptococcus spp., 8% Pediococcus spp.¸7% Lactococcus spp. and 5% of other microbes (Fiorda et al., 2017). Both the bacteria and yeast in the milk kefir grains are bound by kefiran, a water-soluble branched glucogalactan that is formed with glucose and galactose along with 127 hexoses and is shown to exhibit the potential properties of antibacterial, cholesterol regulation and antioxidant activity, making it distinct from other exopolysaccharides (Huang et al., 2013; Rodrigues et al., 2005; Yilmaz-Ersan et al., 2018). Water kefir grain differs in that these types of grains are usually whitish to pale grey, smaller and smoother that look like rock salt with a typical diameter ranging between 0.1 and 2 cm (Fermente Bir İçecek et al., 2017; Guzel-Seydim et al., 2021). Water kefir grains consist of a vast spectrum of microbial communities including 70% Lactobacillus spp., 10% Leuconostoc spp., 10% Acetobacter spp., 5% Bifidobacterium spp., and 5% other microorganisms (Fiorda et al., 2017). The water kefir grains are bound by dextran which is chemically structured by α-D-(1→6)-linked glucopyranosyl residues with (1→3)-linked side chains (Moinas et al., 1980). Although the symbiotic association in both milk kefir and water kefir reveal a commonality in the bacterial species, but the ratio and genera of these microorganisms result in differences in the final beverages (Fiorda et al., 2017). Both of these kefir grains share similar characteristics in that they are reusable for successive fermentation, and the biomass will increase in every cycle of production, but the yield may vary with fermentation variables (Guzel-Seydim et al., 2011). Historically, milk kefir was discovered in the Caucasian region while water kefir’s history remained unclear (Rosa et al., 2017).

Fig. 1.

Graphic illustration on (A) health benefits, preparation and factors affecting microbial diversity in kefir beverages with (B) comparisons of the milk and water kefir

Kefir’s popularity has surged with great momentum along the trend of healthy diet and functional food as it provides an inherent source of boosting immunity defence against viruses (Simova et al., 2002). According to Fortune Business Insight (2020), the kefir market size reached USD 1.23 billion in 2019 and is expected to experience a surge in growth at a Compound Annual Growth Rate (CAGR) of 5.4% by 2027. During the days when clinical treatment was not advanced, kefir has shown to offer potential beneficial health effects and was used as a functional food for treating illnesses or diseases (John & Deeseenthum, 2015). Over the years, milk and water kefirs have gained a reputation for its excellent health effects that include antimicrobial (Powell et al., 2007), probiotics and improving gut health (Garrote et al., 2010), improving lactose digestion (Rattray & O’Connell 2011), anticarcinogenic (Khoury et al., 2014), antioxidant (Chen et al., 2015), regulating cholesterol (García-Burgos et al., 2020) and more recently on controlling blood glucose level (Salari et al., 2021) through evidence of in-vitro and in-vivo researches when taken regularly at serving size typically ranging from 180 mL to 500 mL in a day (Chong et al., 2023; John & Deeseenthum, 2015; Rosa et al., 2017). Research on the relationship between dosage and efficacy has not been well-established, thus further clinical studies are required to identify optimal dosage regimes for certain health effects with consideration of an individual’s age, individual condition and health status. This review provides current insights into kefirs and their recent updates on their health-promoting effects, comparing milk and water kefir for their processing, microbiological composition and current application of exopolysaccharides of milk kefir in the food industry. This information will provide new opportunities for kefir improvement and assist in the development of functional food production.

Health benefits of Kefir

Probiotics and protection in Gastrointestinal tract

The regular intake of kefir can desirably improve composition of gastrointestinal microbiota and immune response of person’s lumen (Rosa et al., 2017). The luminal surface of the gastrointestinal tract is colonised by a wide spectrum of microbial communities, which are responsible for regulating important processes associated with human health (Bellikci-Koyu et al., 2019; Kim et al., 2019a, b). Milk kefir, which is rich in probiotics, may fight against those pathogenic bacteria by binding the epithelial cells in the gastrointestinal tract and releasing bacteriocin and acids that inhibit invading pathogens in the host’s gut. It may also support intestinal mobility in the removal of pathogenic bacteria, producing improvement of microflora balance and a well-maintained digestive system through colonisation of beneficial microbial populations (Bellikci-Koyu et al., 2019; Garrote et al., 2010; Kim et al., 2019a, b; Leite et al., 2013). In a recent study by Dahiya and Nigam (2023), they explained that consuming milk kefir can contribute in supporting the replenishment of gut microbiota and alleviate antibiotic-induced gut dysbiosis, a condition of destabilised gut microbiota balance which causes negative effects such as weight fluctuation, spike of blood sugar and diarrhoea. A study suggested that milk kefir might stimulate the secretion of immunoglobulin A (IgA) in the gut system. The IgA can support immune defence by neutralising pathogens and restricting their adhesion to epithelial cells of the gastrointestinal tract where this mechanism is potentially capable of minimising gastrointestinal infection by hindering pathogen colonisation (Salari et al., 2022). The kefiran, an exopolysaccharide in the milk kefir grains is known to support replenishing of intestinal mucosa and immunity system wing to its strong probiotic characteristics, making it a potential treatment for bowel diseases and gastrointestinal diseases (Fard et al., 2017; Rodrigues et al., 2016).

Water kefir, rich in probiotics, may contribute to improving gastrointestinal homeostasis and support gut health (Hamida et al., 2021). The regular intake of water kefir has been evaluated for showing high effectiveness in supporting intestinal homeostasis and modulating the immune system through the production of short-chain fatty acids including acetate, butyrate and propionate derived from fermentation (Calatayud et al., 2021). Studies suggested that the Bifidobacterium psychraephilum in water kefir exerted a probiotic effect due to its ability to produce acetate, which may facilitate metabolism within the body and improve the immune system (Gulitz et al., 2013; Laureys et al., 2016). Koh et al. (2018) have suggested that Liquorilactibacillus mali K8 in water kefir was resistant towards gastrointestinal stress while Abatemarco Júnior et al. (2018) stated that Lentiactobacillus diolivorans 1Z, a probiotic bacterial strain in water kefir showed high antimicrobial activity towards Salmonella infections. Kefir, which consists of good source of probiotics and bioactive compounds is highly recommended for modulating gut health.

Control of blood glucose level

Diabetes is known as an endocrine disease that is often caused by an abnormal spike in blood glucose level and this disease can lead to dysfunction in the liver or kidneys and loss of muscle and tissue weight (Alihosseini et al., 2017; Aloulou et al., 2012; Bhattacharya et al., 2014). Milk kefir has gained recognition among various functional foods for its potential diabetic control properties (Salari et al., 2021; Alihosseini et al., 2017; Calatayud et al., 2021). Within milk kefir, the lactic acid bacteria may control blood glucose and improve insulin resistance (Salari et al., 2021). A prior animal study examined the impact of milk kefir consumption on blood glucose level, where the Wistar rats with diabetic mellitus administered with milk kefir for a month resulted in a noteworthy drop in plasma glucose compared to the control group (Hadisaputro et al., 2014). In a clinical trial, regular intake of milk kefir by sixty diabetic patients showed a significant decrease in fasting blood glucose and glycated haemoglobin (HbA1C), showing that milk kefir is a promising beverage that can potentially improve glucose regulation of diabetic patients (Ostadrahimi et al., 2015). In another study involving Type 2 Diabetes patients, the consumption of milk kefir consisting of Lactobacillus acidophilus, Lactococcus casei, Streptococcus thermophilus, and Bifidobacterium lactis for two months showed a 2.12-fold decrease in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), which is clinically relevant as it suggests an improvement in insulin sensitivity and potentially can prevent a sudden spike in blood sugar level (Alihosseini et al., 2017). In a recent study by Calatayud et al. (2021), it was shown that Wistar rats fed with water kefir for five weeks resulted a positive improvement in blood glucose levels as compared to the control group. The control of blood glucose may be driven by the presence of probiotic strains, particularly Lentiactobacillus spp. and Bifidobacterium spp. in water kefir that inhibit α-glucosidase and pancreatic α-amylase, reducing postprandial rise in blood glucose level and hence delaying the onset of hyperglycaemia, a high blood glucose level condition in diabetic patients (Calatayud et al., 2021).

Antioxidant

Oxidative stress is generated and accumulated from free radicals within our human body through physiological modulation in various organs, exposure to environmental factors and inappropriate diet which cause substantial damage to biological materials through localised body cells (Chong et al., 2023; Pizzino et al., 2017; Stobiecka et al., 2022). Kefir is acclaimed as a recommended dietary option that can possibly mitigate oxidative stress and damage due to its beneficial microorganisms and fermentation metabolites that serve as excellent radical scavengers (Fiorda et al., 2017). Fermentation of kefir leads to the formation of antioxidants including peptides from milk casein hydrolysate, amino acids such as tryptophan, cysteine, taurine and tyrosine, vitamins A and E, carotenoids, superoxide dismutase, glutathione peroxidase and catalase. These antioxidants can facilitate in halting lipid degradation, muscle contraction and relaxation process, cholesterol metabolism and protein oxidation (Chen et al., 2015; Dufresne & Farnworth, 2000; Liu et al., 2005; Ozcan et al., 2018). Various researchers have demonstrated the increase of antioxidant activity during milk fermentation (Grishina et al., 2011; Satir & Guzel-Seydim, 2015; Yilmaz-Ersan et al., 2016). In the studies conducted by Grishina et al. (2011) and Yilmaz-Ersan et al. (2016), the findings showed that milk kefir consisted of approximately three times higher amounts of phenolic compounds as compared to unfermented milk. Their findings suggested that proteolysis induced by microflora within targeted protein fractions has triggered the release of amino acids. Liu et al. (2005) and Yilmaz-Ersan et al. (2016) also measured the increasing trend of antioxidant activity during fermentation process of milk kefir using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, a test used to quantify a substance’s ability to neutralise free radicals. In the shelf-life test, the scavenging ability of milk kefir was shown to sustain in storage for at least three weeks (Ozcan et al., 2018; Patil et al., 2021; Yilmaz-Ersan et al., 2016). Güven et al. (2003) and Ozcan et al. (2018) explained that the excellent scavenging power of milk kefir was attributed to microbial activity and phenolic compounds specifically rich in vitamin E and β-carotene, suggesting a decrease in the likelihood of sudden coronary thrombosis, atherosclerosis, and gastric lesions driven by oxidative damage.

Water kefir also exhibits an excellent scavenging effect. Kumar et al. (2021) have revealed the strong neuroprotection and antioxidant ability of water kefir against hydrogen peroxide induced oxidative stress in neuroblastoma cells (SH-SY5Y) which was often used as a model to study neurodegenerative disease, and the effects were mediated through the improvement of superoxide dismutase, catalase and apoptotic genes regulation in preventing oxidative stress. The scavenging capability in water kefir fermented in kiwi and pomegranate juice was attributed to a wide range of polyphenols such as ellagic acid, anthocyanin, and punicalagin isomers serving as oxygen scavengers, which are responsible in preventing oxidation damage in body cells (Randazzo et al., 2016). The lactic acid bacteria, organic acid and enzymes present in water kefir were found to improve the antioxidant power (Sabokbar & Khodaiyan, 2016). For example, β-glycosidase, a fermentative enzyme from fermentative microbes in water kefir can break down phenolics into simpler forms which can contribute to the increase in total phenolic content (Sabokbar & Khodaiyan, 2016). L. acidophilus, a lactic acid bacteria species found in water kefir grain, was found to exert a positive effect on the DPPH radical capability (Sabokbar & Khodaiyan, 2016). In an animal study, Brasil et al. (2019) reported that the mice group fed with water kefir for two weeks led to a significant increase in antioxidant activity, catalase enzyme reactivity and superoxide dismutase, significantly reducing the proteolysis activity. It was also believed that drinking water kefir can potentially prevent the emergence of gastric lesions against ethanol ulcer model by retaining the scavenging ability in the tissue within the stomach (Brasil et al., 2019). Water kefir was also proven to exhibit a high potency to reduce ferricyanide complex to ferrous form, which revealed its high reducing power and as an established indicator of antioxidant capability (Jawfi et al., 2012).

Antimutagenic and anticarcinogenic

The effect of kefir supplementation on its potential in reducing risk of cancer has gained huge popularity and has been extensively explored in the clinical and nutraceutical industries (Guzel-Seydim et al., 2021; Hatmal et al., 2018). According to Moreno et al. (2007), milk kefir supplementation might stimulate the activation of CD4⁺ T cell activity which is responsible for modulating production of cytokines, small protein molecules that serve significant roles in suppressing inflammatory conditions and activating macrophages for inhibiting growth of tumour cells. Additionally, the presence of polysaccharides and bioactive components in milk kefir, including protein, peptides, polyphenols, organic acid, genistein and vitamins, can favourably hinder the onset of cancer or prevent the development of tumours by halting specific enzyme action, thereby preventing the occurrence of carcinogenesis process (Ahmed et al., 2013; Guzel-Seydim et al., 2021; Hatmal et al., 2018; Leite et al., 2013; Rosa et al., 2017). Research conducted by Khoury et al. (2014) and Hosona et al. (1990) has revealed that the bacterial strains isolated from milk kefir exhibited a high antimutagenic activity up to 98.5% as they bind to mutagens and are excreted with faeces to maintain the homeostasis of the colon and prevent colon cancer. Hosono et al. (1990) also proposed that milk kefir could possibly inhibit the occurrence of cell proliferation and initiate apoptosis, a process of programmed cell death in HT 29 and Caco-2 colorectal cancer cells. Hosono et al. (1990) described that milk kefir could halt the progression of cell cycle at G1 phase, hinder mRNA expression of transforming growth factor-α (TGF-α) and growth factor-β1 (TGF-β1) in the HT 29 cell, indicating that that milk kefir consumption could reduce the risk of colon cancer from growing. In the Ames test, a biological assay assessing mutagenicity using isolated bacteria in food system from chemicals causing DNA mutation, milk kefir was able to lower the mutagenicity rate of methyl methanesulfonate and sodium azide by a difference of 25% and 21% respectively, as compared to unfermented milk, suggesting the potential of milk kefir preventing DNA damage caused by mutagens (Guzel-Seydim et al., 2006). This can be achieved by the production of conjugated linoleic acid isomers, palmitoleic acid, butyric acid, oleic acid and palmitoleic acid in milk kefir.

Water kefir was identified to possibly induce apoptotic cell death and exhibit neuroprotective effects by stimulating the superoxide dismutase and catalase activity (Kumar et al., 2021). Superoxide dismutase is an enzyme that is responsible for suppressing the growth of pancreatic cancer, and catalase can protect normal cells from mutations (Wilkes et al., 2017). The anticancer effect of water kefir was also proposed by Koh et al. (2018) who mentioned that water kefir could hinder breast cancer cell growth in both in-vitro and in-vivo studies, primarily by the action of apoptosis, immunomodulation and trigger of cytotoxic T cells activity to remove mutagenic and cancerous cells. In an animal study, the finding suggested that L. casei BL23 found in water kefir could positively resist the proliferation of colorectal cancer cells through lowering the histological scores and proliferative values (Jacouton et al., 2017).

Regulating cholesterol

Hypertension is characterised as abnormal spike in the blood pressure level within the arteries which can lead to severe complications such as cardiovascular diseases, kidney diseases and stroke (Carey et al., 2018). National Health and Morbidity Survey (NHMS) indicated patients suffering from hypertension in Malaysia have risen to 30% in 2019 and became one of the most significant public health problems (Carey et al., 2018; Zaki et al., 2021). Drinking kefir has been suggested as a way to help regulating blood pressure and cholesterol (Maeda et al., 2004; Vujičić et al., 1992). The conjugated linoleic acids can potentially reduce cholesterol and suppress atherogenesis (Guzel-Seydim et al., 2006). The peptides of milk kefir can lower blood pressures by inhibiting angiotensin I-converting enzyme (ACE) activity, the cause of high blood pressure as it continually degrades bradykinin which stimulates vasodilation and speeds up the production of angiotensin ll hormone causing a sharp increase in blood pressure (Möller et al., 2008). Research has identified supporting evidence of regulation of serum cholesterol concentration through two mechanisms, i.e. boosting synthesis of new bile acids which facilitates fats breakdown and possibly toxic removal in the body or potentially lowering cholesterol solubility which reduces cholesterol absorption in blood stream (Begley et al., 2006; Brashears et al., 1998). In an animal study, Liu et al. (2005) found that the cholesterol mice group consuming soymilk kefir had a lower total cholesterol and LDL level as compared to the control group, which may be driven to presence of lactic acid bacteria that contribute to controlling blood pressure including L. kefiranofaciens and L. delbrueckii. Given that milk kefir is a representative of lactic acid bacteria-yeast fermented milk, it is a promising candidate for supporting cardiovascular health through its potential function of metabolising cholesterol and minimising resorption from the intestinal lumen (García-Burgos et al., 2020).

Drinking water kefir has also been associated with the potential of controlling cholesterol level. In the animal studies, Alsayadi et al. (2014) revealed a significant improvement in the lipid profile in Wistar rats that were fed with water kefir for five weeks meanwhile Chen et al. (2018) observed a decrease in body weight and hepatic lipid accumulation in Dawley rats fed with the water kefir consisting of Liquorilactobacillus mali APS1 isolated from water kefir grain. Rocha-Gomes et al. (2018) had found that mice group treated with water kefir resulted a lower total cholesterol and LDL level as compared to milk kefir-treated mice, suggesting that water kefir is more effective in controlling the cholesterol profile.

Antimicrobial

Milk kefir can exhibit a strong antimicrobial resistance towards pathogens as supported by a wide range of beneficial microbial communities and fermentation metabolites such as lactic acid, hydrogen peroxide, carbon dioxide, acetaldehyde, and bacteriocins (John & Deeseenthum, 2015). Previous research identified that milk kefir comprised of 3.5 kDa bacteriocin ST8KF and hydrogen peroxide produced by Lactiplantibacillus plantarum subsp. plantarum, serving as important antibacterial metabolites (Powell et al., 2007). Yüksekdag et al. (2004) revealed that Turkish milk kefir consisted of 21 lactic acid bacteria isolates, particularly Lactococci strains, producing hydrogen peroxide ranging between 0.04 and 0.19 µg/ml. Notably, all these Lactococci strains were evaluated to exhibit high inhibitory activity against Staphylococcus aureus, a food pathogen that is associated with food poisoning. Santos et al. (2003) examined the ability of Lactobacillus acidophilus and Lactobacillus kefiranofaciens isolated from milk kefir to inhibit various pathogens including Shigella flexneri, Salmonella typhimurium, Escherichia coli and Yersinia enterocolitica effectively. Numerous studies revealed that kefiran showed strong antibacterial and antifungal effects against certain unicellular bacterial species including Streptococcus faecalis KR6 and Fusarium graminearum CZ1 and also filamentous fungal species such as Aspergillus flavus AH3 (Diniz et al., 2003; Ismaiel et al., 2011; Kwon et al., 2003; Rodrigues et al., 2005). Their finding was consistent with Medrano et al. (2008), who assessed an increasing trend of inhibitory activity of kefiran against Bacillus cereus B10502 when administered at a dosage increasing from 300 to 1000 mg/L. Through in-vitro studies, a positive relationship between milk kefir’s antimicrobial properties and health benefits was found specifically in treating vaginal infection caused by Gardnerella vaginalis (Afifi et al., 2020) and also in preventing gastrointestinal infection caused by Salmonella bacteria (Gut et al., 2022). As promising evidence is derived mostly from in-vitro research where further studies involving controlled clinical trials are necessary to evaluate the efficacy of kefir intake in the prevention and treatment of infectious diseases.

Similar to milk kefir, several studies found that the production of organic acids in water kefir such as lactic acid and acetic acid, will lead to an acidic environment which can suppress the growth of pathogens such as Salmonella spp., Escherichia coli, Aspergillus flavus and Staphylococcus aureus (Fiorda et al., 2016; Gamba et al., 2015; Romero-Luna et al., 2020; Zavala et al., 2016). The cell-free supernatants in water kefir have demonstrated fungicidal resistance towards Aspergillus parasiticus at the pH of 4.55 and no aflatoxin was detected (Gamba et al., 2015). More recently, Çevik et al. (2019) found that brown sugar was more effective in inhibiting pathogens such as Staphylococcus aureus, Shigella sonnei, Escherichia coli and Candida albicans as compared to molasses and demerara sugar.

Improve lactose digestion

Lactose intolerance is a reaction when the human body is unable to digest lactose due to the insufficient amount of lactase, an enzyme that is responsible for metabolising the lactose (John & Deeseenthum, 2015). During milk kefir fermentation, yeast will metabolise lactose into fermentation metabolites, mainly lactic acid, ethanol, carbon dioxide and acetic acid (Ganatsios et al., 2021; Rattray & O’Connell, 2011). Lactose can be broken down through two different metabolic pathways, i.e. lactose permease system (LPS) and phosphoenolpyruvate-dependent phosphotransferase system (LPPDPS) (Harutoshi, 2013; Sharma et al., 2023). The LPS pathway involves the metabolism of lactose into glucose and galactose, while LPPDPS breaks down lactose into glucose and galactose-6-phosphate through the hydrolysis activity by β-galactosidase enzyme (Harutoshi, 2013; Sharma et al., 2023). The lactic acid bacteria will then proceed to the two pathways, including homofermentative metabolism predominantly produces lactic acid while the heterofermentative pathway produces various of metabolites such as ethanol, carbon dioxide and volatile acids (Alves et al., 2021; Harutoshi, 2013; Sharma et al., 2023). As a result, the hydrolysis of lactose by lactic acid bacteria in milk kefir can improve lactose digestion and absorption. Studies by Hertzler and Clancy (2003) and Labayen et al. (2001) found that commercial milk kefir could exhibit similar effectiveness as yoghurt in improving lactose tolerance. Rattray & O’Connell (2011) have examined that the microbial community in milk kefir consisted of a certain level of β-galactosidase activity which was responsible for breaking down lactose in the milk into simpler sugars that were readily absorbed in the system. Alm (1982) stated that milk kefir’s lactose was reduced by 30% as compared to unfermented milk.

As water kefir is made by fermenting a sucrose-containing solution or a non-dairy beverage such as fruit or vegetables, this product is an ideal and healthier option for lactose-intolerant consumers, vegans or individuals who have severe allergic reactions towards dairy food (Guzel-Seydim et al., 2021). Despite the controversy or gap in understanding of whether milk kefir is effective in improving lactose digestion due to the fact it is not lactose-free beverage (Pražnikar et al. 2020), study conducted by Hertzler and Clancy (2003) found that milk kefir consumption can reduce the degree of flatulence by 71% while De Vrese (1992)’s finding that the level of β-galactosidase activity induced by microorganism in milk kefir is 60% higher than in yogurt, provided possible explanation that intestinal absorption of lactose was improved. In recent years, numerous researchers have published studies and warrant that milk kefir can improve lactose absorption (Ganatsios et al., 2021; John & Deeseenthum, 2015; Rosa et al., 2017; Sharma et al., 2023).

Processing of Kefir

Processing of kefir is relatively simple and has been culturally practiced for centuries. Milk and water kefir can be processed through three main methods including traditional, backslopping and direct-to-vat inoculation (Beshkova et al., 2002; Leite et al., 2013; Rattray & O’Connell, 2011). Each processing method has its own set of advantages and disadvantages in terms of source of kefir grain, product quality, scalability and microbial diversity and is presented in Table 1. The selection of processing method ultimately depends on the intended application, whether it is for artisanal or commercialisation purposes. The direct-to-vat method is used in large scale productions supplying kefir for commercialisation. Figure 2 shows a general process of kefir-making following a smaller production scale, i.e. bench-top production where the traditional or backslopping is preferred. Firstly, the substrate is subjected to pasteurisation at 95 °C for 15 min to improve bioavailability of nutrients for those fermenting lactic acid bacteria (Sarkar, 2008). After cooling down, the kefir grains are added to the substrate in a partially sealed glass container and proceed for fermentation (Yilmaz et al., 2022). At the end of fermentation process, kefir grains are filtered and separated from the milk or water kefir by filtration. Following fermentation, milk kefir undergoes ripening process where it is stored in refrigerated conditions for 8 to 24 h for a more viscous, tangy and slight effervescence final product (Laureys et al., 2018). Unlike milk kefir, water kefir is typically subjected to a secondary fermentation instead of ripening and this process involves bottling of water kefir with additional fruits, resulting a clear, fizzy and slightly acidic fermented fruit beverage. As the quality of kefir can be driven by factors such as fermentation temperature, time, types of kefir grains, substrate availability, more research studies on kefir production are needed to further validate and determine the exact parameters for producing a consistent and standard kefir.

Table 1.

Key differences of three processing methods: traditional, backslopping and direct-to-vat inoculation.

(adapted from Gentry et al., 2023; Kim et al., 2018; Nejati et al., 2020)

Methods	Source	Advantages	Disadvantages
Traditional	Natural kefir grains	• Preserves natural and high diversity of microbes • Produces a kefir with well-bodied flavour and potential health promoting characteristics due to wide range of probiotics	• Less controlled • Inconsistencies in microbial composition • Labour intensive, making it not suitable for industrial use
Backslopping	Percolate from previous batch	• Cost-effective • Suitable for small-scale production • High adaptability of microbial population during fermentation	• High possibility in shifting of microbial composition • Risk of contamination when not properly controlled
Direct-to-vat inoculation	Freeze-dried kefir grains with isolated microbial strains	• Controlled fermentation and high reproducibility of microbial composition • Produces a consistent, stable and safe kefir product • Improves productivity and suitable for industrial production	• Limited probiotics diversity • Potentially minimised bioactivity and sensory complexity • High cost

Fig. 2.

A typical kefir production steps

Microbiological composition

Kefir consists of a wide array of microorganisms, predominantly derived from lactic acid bacteria and yeast (Verruck et al., 2019). Nevertheless, the microbial composition of kefir is strongly influenced by environmental condition of kefir grains origin, processing parameters and substrate availability which influence the probiotic strains associated with health benefits (Ilıkkan & Bağdat, 2021; de Sainz et al., 2020). The variations in the dominant bacterial and fungal species, fermentation kinetics and metabolites have led to commercial kefirs vary in final microbiological composition, sensory properties and health-promoting characteristics. Each lactic acid microorganism contributes distinct role in fermentation. For instance, Lactobacillus kefiranofaciens has been associated with the improvement of viscosity during fermentation while Leuconostoc mesenteroides is responsible in aroma formation that eventually affected the final sensory quality of kefir (Dailin et al., 2016; Endo et al., 2002). The health promoting characteristics contributed by lactic acid bacteria in kefirs from different geographical locations such as Brazil, Ireland, Malaysia, Iran, Australia and Argentina have been investigated extensively and well-documented with the approach of Next Generation Sequencing, an advance metagenomic technique that allows comprehensive taxonomic identification and profiling (presented in Tables 2 and 3). Both kefirs differ significantly in their microbial community and diversity. Milk kefir is enriched with a nutrient-dense environment due to protein and fat matrix. It supports a more complex and wider spectrum of microbial communities and harbours a diverse bioactive compound, offering a more pronounced health benefits and sensory profile. In contrast, water kefir, a dairy-free and lighter option presents a simpler microbial community as compared to milk kefir.

Table 2.

Types of lactic acid bacteria and its functional properties in milk kefir from various geographical locations

Lactic acid bacteria	Functionalities	References	Locations
Lb. kefiri	3.1 Probiotics and protection in gastrointestinal tract	Dobson et al. (2011) Leite et al. (2013)	Ireland Brazil Indonesia Australia
		Yusuf et al. (2020)
		Alraddadi et al. (2023)
Lc. mesenteroides	3.2 Control of glucose level: Lower the risk of obesity and improving plasma insulin resistance	Leite et al. (2013) Sindi et al. (2020) Dobson et al. (2011)	Brazil Britian Caucasus Ireland
Lb. kefiranofaciens	3.2 Control of glucose level 3.4 Anticarcinogenic	Garofalo et al. (2015) Dobson et al. (2011) Walsh et al. (2016) Zamberi et al. (2016)	Bosnia Ireland France Malaysia
Lb. helveticus	3.3 Antioxidant effect	Garofalo et al. (2015) Dobson et al. (2011) Sindi et al. (2020)	Italy Ireland Britain Caucasus
Lc. Lactis	3.4 Anticarcinogenic: Promoting cytotoxicity towards tumour cells	Garofalo et al. (2015) Ilıkkan and Bağdat (2021) Leite et al. (2013)	Bosnia Turkey Brazil
Lb. acidophilus	3.5 Regulating cholesterol 3.6 Antimicrobial: Produce bacteriocin	Bourrie et al. (2021) Sindi et al. (2020) Dobson et al. (2011)	Canada Britain Caucasus Ireland
Lb. crispatus	3.6 Antimicrobial: Prevent viral infection	Garofalo et al. (2015) Zamberi et al. (2016)	Italy Malaysia
Lb. buchneri	3.7 Improve lactose digestion and tolerance	Ilıkkan and Bağdat (2021) Leite et al. (2013)	Turkey Brazil

Functional properties have been numbered following health benefits outlined in above section

Table 3.

Lactic acid bacteria and its functional properties in water kefir from various geographical locations

Lactic acid bacteria	Functionalities	References	Locations
Lb. parabuchneri	3.1 Probiotics and protection in gastrointestinal tract	Magalhães et al. (2010)	Brazil Turkey Argentina
		Yerlikaya et al. (2022)
		Rodríguez et al. (2023)
Lb. plantarum	3.1 Probiotics and protection in gastrointestinal tract	Angelescu et al. (2019)	Romania Argentina
		Gamba et al. (2021)
Lb. paracasei	3.1 Probiotics and protection in gastrointestinal tract 3.4 Anticarcinogenic 3.6 Antimicrobial	Kim et al. (2019a, b) Magalhães et al. (2010) Mousavi Jam et al. (2021)	Korea Brazil Iran
Lb. harbinensis	3.3 Antioxidant 3.4 Anticarcinogenic: Producing high phenolic compound	Angelescu et al. (2019) Talib et al. (2019)	Romania Malaysia
Lb. hilgardii	3.4 Anticarcinogenic and antimutagenic	Pourramezan et al. (2020) Zanirati et al. (2015)	Iran Brazil Malaysia
		Kumar et al. (2021)
Lb. statsumensis	3.4 Anticarcinogenic	Fiorda et al. (2016)	Mexico
Lb. nagelii	3.5 Regulating Cholesterol	Gulitz et al. (2011) Laureys et al. (2018c) Zanirati et al. (2015)	Germany Belgium Brazil

Functional properties have been numbered following health benefits outlined in above section

In research on kefir production, the main lactose-fermenting yeasts such as Candida kefyr and Kluyveromyces marxianus ssp. Marxianus were detected abundantly in milk kefir, while Saccharomyces cerevisiae, Hanseniaspora and Guehomyces were the dominant yeasts identified in water kefir (Diosma et al., 2014; Garofalo et al., 2015; Guzel-Seydim et al., 2021). Lynch et al. (2021) observed that the Saccharomyces group was predominantly detected in water kefir while the Candida group in milk kefir. Both of these yeast species serve different roles in fermentation where the Saccharomyces group is involved synthesis of complex B vitamins and serve important roles in alcohol and carbon dioxide production which was also widely applied in other products such as wine, cocoa and biofuel while the Candida group built a symbiotic relationship with lactic acid bacteria, serving as a nitrogen and carbon source for synthesis of kefiran for textural improvement of milk kefir (Kieliszek et al., 2017; Parapouli et al. 2002).

A successful kefir fermentation is driven by the metabolism interaction between LAB and yeast (Smid & Lacroix, 2013). Numerous researchers have identified the co-existence and symbiotic relationship between LAB and yeasts in fermented food such as sourdough (Sieuwerts et al., 2018), soy sauce (Feng et al., 2023), wine (Kang et al., 2024) and cheese (Melkonian et al., 2023). Both LAB and yeast exchange nutrients such as vitamins and nitrogen for growth and also molecular signals (Adesulu-Dahunsi et al., 2020; Canon et al., 2021). Stadie et al. (2013) have investigated an enhanced growth in the co-cultivation of LAB strains including Lb. hordei and Lb. nagelii and yeast strains involving Z. florentina and S. cerevisiae when compared with single cultivation. Certain yeast species have been identified to exhibit enzymatic activities and these functionalities lead to the breakdown of milk protein and improve the bioavailability of essential bioactive peptides or amino acids (Cruz-Casas et al., 2021). In return, acid metabolism by LAB has been observed to provide an acidic environment which supports the growth of acidophilus yeast species especially Saccharomyces cerevisiae which is responsible for aroma and taste development (Sieuwerts et al., 2018). These positive microbial interactions involving LAB and yeast will enhance fermentation kinetics and also sensory, shelf life, nutritional and probiotic characteristics (Adesulu-Dahunsi et al., 2020).

Industrial application of Kefiran products

Kefiran is an exopolysaccharide bound to the milk kefir grains and has receiving a great interest among various industries due to its versatility and generally recognised as safe.Thoreux and Schmucker (2001) have found that kefiran could serve as a binding agent, fat replacer, emulsifier, thickening agent, stabilizer, gelling agent, and water binding in the food industry. Interestingly, the incorporation of kefiran as an additive in food products can enhance their textural properties such as viscosity. Besides, a prior experiment has shown that kefiran exhibited an excellent water absorption capability, stability in mixing and wheat dough formation time, which provided a choice of natural additives for bakeries (Fard et al., 2017). Another research has also identified the potential of kefiran as a gelling agent, showing that the addition of kefiran at low temperatures could enhance the viscosity and viscoelastic of acid gel in skim milk (Rimada & Abraham, 2006; Soleimanifard et al., 2015). Kefiran gels are colourless and possess great strength of adhesion due to their high-water binding capability. With that, it is characterised as a good thickener choice in the dairy industry. Moreover, kefiran can also be utilised in frozen confectionery whereby the texture (flow behaviour) can remain unchanged during the freeze-thaw cycle because of its stabilising characteristics (Exarhopoulos et al., 2018). Compared to other food polysaccharides such as dextran and xanthan gum, kefiran stands out due to its additional potential health promoting characteristics. Existing research has suggested that kefiran intake has led an improvement in serum total cholesterol as observed in a study of high-cholesterol rabbits (Uchida et al., 2010).Medrano et al. (2011) have indicated that kefiran with strong probiotic effects can help to maintain a good gut health through balancing of gut microbiota. The strong mechanical stability of kefiran makes it an excellent biodegradable film for packaging and also an encapsulation material for biopharmaceutical drug delivery, providing a more sustainable solution in both the packaging and pharmaceutical industries (Fard et al., 2017; Sabatino et al., 2020; Tan et al., 2020). Hence, kefiran with its excellent physical and health characteristics have made it a potential candidate for food, biopharmaceutical and packaging industries.

Conclusion and future perspective

The development of milk or water kefirs as functional beverages are brought upon by fermentation action of kefir grains, respectively in a milk-based or sucrose containing liquid. Although both milk and water kefir are processed differently, both kefirs are well-acknowledged of their highly nutritious and valuable health benefits including probiotics that help to improve gut health, control of glucose and cholesterol, prevent oxidative damage, inhibit pathogenic microorganisms, improve lactose digestion and reduce risk of cancer. The microbial communities of both kefirs vary significantly according to the types of substrates and origin of kefir grains giving both kefirs their distinctive sensory and functional properties. Milk kefir, supported by its nutrient-dense matrix, foster a higher diversity of beneficial microorganisms, harbouring a diverse fermentation metabolites profile that offers a more comprehensive health promoting characteristics. Water kefir, being a lighter and dairy-free fermented beverage consist of a simpler microbial community and offers an alternative option for those who are lactose intolerance or vegans. It has more targeted health benefits of regulating cholesterol level. The food chain of kefir offers ultimate gain for low-waste and energy efficient production.

Author contributions

Joyce Jen Li Lim: Data curation, Formal analysis, Investigation, Validation, Writing-original draft. Nyuk Ling Chin: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing- review and editing. Chun Wie Chong: Resources, Supervision, Validation, Visualization, Writing- review and editing. Adiratna Mat Ripen: Software, Supervision, Validation, Visualization, Writing- review and editing. Syahmeer How: Supervision, Validation, Visualization, Writing- review and editing Sze Qi Teoh: Data Curation, Investigation.

Declarations

Conflict of interest

The authors declare no potential conflict of interest.

Ethics approval

This article does not require IRB/IACUC approval because there are no human and animal participants.