Lacticaseibacillus compositions and uses thereof

Abstract

The disclosure relates to methods and uses of at least one strain of Lactobacillus paracasei for reducing and / or preventing weight gain in a subject. The disclosure further relates to methods and uses of at least one strain of Lacticaseibacillus paracasei in the prevention and / or amelioration and / or treatment of metabolic syndrome. The present invention further relates to methods and uses of at least one strain of Lacticaseibacillus paracasei in improving cardiometabolic health and / or cardiovascular health.

Description

[0001] LACTICASEIBACILLUS COMPOSITIONS AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] The disclosure relates to methods and uses of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject. The disclosure further relates to methods and uses of at least one strain of Lacticaseibacillus paracasei in the prevention and / or amelioration and / or treatment of metabolic syndrome. The present invention further relates to methods and uses of at least one strain of Lacticaseibacillus paracasei in improving cardiometabolic health and / or cardiovascular health.

[0004] BACKGROUND

[0005] There is a wide market for products that can assist with preventing and / or reducing weight gain or assist with losing weight.

[0006] Obesity is a pathological state characterized by abnormal or excessive fat accumulation caused by a chronic imbalance between energy intake and energy expenditure. Obesity is defined as an increased body weight when compared to height. An individual is considered obese when the body mass index (BMI) level is 30 or higher. The cause of weight gain is essentially excess energy intake and low energy expenditure.

[0007] Obesity is a global health issue and is more prevalent now than ever in both adults and children and many factors come into play such as unhealthy eating habits of food with high sugar and fat content, lack of physical activity and other secondary factors such as genetic, age, gender, behaviour, lifestyle, environment and socioeconomic factors. Obesity is a health issue of significant concern as it can lead co-morbidities, such as metabolic syndrome, hypertension, hypercholesterolemia, type II diabetes, cardiovascular disease, cardiometabolic disease, cancer and raise the overall morbidity in obese individuals putting the healthcare system under economic pressure with significant increasing in obesity- related spending each year.

[0008] The golden standard for losing weight is through diet and exercise. Various diets can be recommended such as low carbohydrate-diet, low fat-diet, Mediterranean diet, etc., no single diet has proven to be superior over the others in term of reducing weight gain, as long term adhesion to a healthier eating plan is the most crucial factor for successful weight management. Physical activity is important for reducing or preventing weight gain as it facilitates burning excess energy from the food intake.

[0009] In order to adhere to a healthier diet and exercise routine, an individual will have to make permanent lifestyle changes and stick to them, which can be very challenging. It is therefore relatively common to relapse and gain weight again after a period of time on a healthy diet and exercise plan. For example, if an individual reverts to their former eating style and / or a more sedentary lifestyle. This is the major pitfall of diet and physical exercise as solution for reducing or preventing weight gain in obese individuals.

[0010] Changing eating and activity habits has proven to be extremely difficult, as it requires changing habits and a permanent commitment to be successful and achieve a long-lasting weight loss, which will lead to a healthier life.

[0011] In addition, the aging process affects lipid metabolism, which can lead to weight gain. Lipid turnover in fat tissue may decrease, making it easier to gain weight, even if individuals do not consume more food or reduce their exercise levels. Hormonal changes during and following menopause can lead to weight gain. People who quit smoking often experience weight gain following cessation of the habit. Furthermore, individuals may wish to reduce either body weight and / or weight gain for cosmetic reasons.

[0012] When diet and physical exercise is not successful, pharmacotherapy can be involved as a second line management of obesity, with multiple drugs on the marked to reduce or prevent weight gain. Lastly, surgery can be considered if an individual is extremely obese and no success with diet, exercise of pharmacotherapy has been achieved.

[0013] Modulating the gut microbiota may be an alternative (or additional) resolution to reduce and / or prevent weight gain. Gut microbiota is a collection of microorganisms residing in our intestine which plays an important role in nutrient utilization, metabolism, barrier protection and immunological processes.

[0014] Obese and overweight individuals have been shown to have a specific gut microbiota, characterized by dysbiosis or imbalance and lower microbial diversity compared to individual with normal weight. A decrease in some bacterial phyla, such as the relationship between Bacteroidetes and Firmicutes, with lower proportion of Bacteroidetes and higher proportion of Firmicutes compared to individuals without obesity have been shown to facilitate energy extraction from the ingested food and increased energy storage in adipose tissue. Further, altered microbiota leads to a supressed production of fasting-induced adipose factor, causing increased storage of triglycerides in adipose tissue and low release of appetite hormones such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), promoting food intake. Thus, gut microbiota is important for maintaining homeostasis of energy metabolism.

[0015] Lacticaseibacillus species are a part of the gut microbiota and is a collection of probiotic strains with the ability to balance the gut barrier integrity and mucosal barrier defence, promote anti-inflammatory responses and ameliorate host immune responses. Modulating gut microbiota metabolism through dietary supplementation of strains of Lacticaseibacillus has been shown to be an effective strategy to prevent various diseases and disorders such as asthma, inflammatory bowel disease (IBD), cardiovascular disease, neuroinflammatory disease, metabolic syndrome, osteoporosis and chronic infections in patients.

[0016] Therefore, modulation of gut microbiota though probiotic dietary supplementation may be an attractive strategy for reducing and / or preventing weight gain.

[0017] There is an unmet need in the field for efficient methods for reducing and / or preventing weight gain.

[0018] Hence, it would be advantageous to provide a method for reducing or preventing weight gain by administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0019] Cardiometabolic syndrome is a constellation of maladaptive cardiovascular, renal, metabolic, prothrombotic and inflammatory abnormalities, which individually and interdependently leads to a substantial increase in morbidity and mortality globally.

[0020] Despite a complex interaction between genetic susceptibility and lifestyle factors in the pathogenesis of cardiometabolic diseases, dietary prevention is considered as a cornerstone in the management of cardiometabolic syndrome since it ranks as the top modifiable risk factor for cardiometabolic health.

[0021] Gut microbiota is a collection of microorganisms residing in our intestine which plays an important role in nutrient utilization, metabolism, barrier protection and immunological processes. Recently, mounting evidence from both preclinical models and human studies demonstrates a close link between changes in gut microbiota and the development of cardiometabolic disorders through several mechanisms, such as increased production of lipopolysaccharide, disturbance in gut immune signalling, and perturbation in the metabolism of bile acid, short-chain fatty acids, trimethylamine-N-oxide, and uremic toxins. Therefore, modulation of gut microbiota has been regarded as a novel strategy for the treatment and prevention of cardiometabolic diseases. Among currently available strategies, probiotic supplementation, especially Lactobacillus and Bifidobacteria, is the most widely studied and attractive approach in practice.

[0022] Lacticaseibacillus paracasei (L. paracasei) is a homofermentative species of lactic acid bacteria that are commonly used in dairy product fermentation and as probiotic cultures. Several strains from L. paracasei have demonstrated a favourable effect on host metabolism. For example, L. paracasei strain NL41 alleviated insulin resistance in a rat model of diabetes mainly through suppressing oxidative stress and inflammation and restoring pancreatic beta cell functions. Similarly, supplementation of L. paracasei strains was also reported to effectively slow down body weight gain and reduce fat accumulation in mice exposed to Western diet.

[0023] Additionally, L. paracasei strain 8700:2 was reported to be able to alleviate gut permeability and translocation, and enhance immunologic defence against known pathogens and viral infections. Given that gut permeability and immunity responses are closely involved in the progression of cardiometabolic diseases, we hypothesize that L. paracasei may function as a feasible dietary supplementation for the prevention of cardiometabolic disorders. However, no pre-clinical models could fully reproduce human cardiometabolic diseases and its multifactorial characteristics with high fidelity. Therefore, in this study, we aimed to evaluate the effect of L. paracasei in a primary care-based, randomized, double-blind and placebo-controlled trial of 130 healthy subjects at high risk for cardiometabolic diseases, with 12 weeks of daily L. paracasei supplementation.

[0024] Modulating microbial metabolism via probiotic supplementation has been proposed as an attractive strategy for the prevention of cardiometabolic diseases. Recently, Lacticaseibacillus paracasei (L. paracasei) was reported to alleviate metabolic disorders in murine models, however, its beneficial effects in humans remain to be determined. This study evaluated whether L. paracasei supplementation could improve endothelial function and cardiometabolic health in subjects with metabolic syndrome (MetS).

[0025] In this randomized, double-blind and placebo-controlled trial among 130 participants with MetS, but otherwise healthy subjects, were randomly assigned to placebo or L. paracasei (10 billion CFU) daily for 12 weeks. Endothelial function was measured by flow-mediated slowing, and cardiometabolic health was determined by both components and severity of MetS. Ideal compliance was defined as consumption no less than 70% of the capsules. The endothelium is an active inner layer of the blood vessel and is the key regulator of vascular homeostasis. Endothelial dysfunction is the earliest detectable deterioration of vessel wall and precedes the development of morphological atherosclerotic changes and contributes to the clinical events. Importantly, as endothelial dysfunction is reversible with treatment of risk factors, it is regarded as a potential therapeutic target and measure of efficacy of preventive strategies.

[0026] Several dietary approaches have been developed to delay the deterioration of endothelial function through modulating gut microbiota. For example, consumption of aronia berry (poly)phenol for 12 weeks improved arterial function in prehypertensive adults, through increasing the richness of gut microbiota and promoting the abundance of butyrate- producing species. Another randomized placebo-controlled study also found that supplementation of fish oil-derived long-chain monounsaturated fatty acids improved endothelial function through enhancing the production of short-chain fatty acids, which in turn stimulated the secretion of glucagon-like peptide-1.

[0027] There is an unmet need in the field for efficient methods for modulating endothelial functions. The inventors have surprising found that 12-week supplementation of L. paracasei 8700:2 significantly improved endothelial function, with a more prominent beneficial effect in those with worse baseline endothelial functions.

[0028] Several mechanisms have been implicated in the pathogenesis of endothelial dysfunction, such as oxidative stress, inflammation, dyslipidaemia characterized by high levels of LDL- c and low levels of HDL-c, hypertension, and insulin resistance. Mechanistically, remnant cholesterol particles were reported to accelerate endothelial dysfunction through inducing endothelial progenitor cells senescence and suppressing endothelial nitric oxide synthase activity. Moreover, mounting evidence from prospective cohorts demonstrated that levels of remnant cholesterol served as an independent and residual risk factor for the development of cardiovascular diseases. Therefore, remnant cholesterol has been considered as a novel therapeutic target for cardiometabolic diseases and lots of efforts have been made in recent years to explore effective agents to lower remnant cholesterol. However, currently existing lipid-lowering agents, either alone or in combination, lead to a minor reduction in remnant cholesterol and no drugs specifically targeting remnant cholesterol has been approved by regulatory authorities.

[0029] Hence, there is an unmet need in the field for identifying substances that can target remnant cholesterol and are safely tolerated. SUMMARY

[0030] The inventors have surprising identified that L. paracasei administration lowered remnant cholesterol with notable between-group differences regardless of protocol deviation and its baseline level. The findings provide evidence that intake of L. paracasei (e.g. on a daily basis) may be an attractive approach to lower remnant cholesterol and its associated cardiovascular risk.

[0031] The disclosure relates to a method for reducing and / or preventing weight gain in a subject. In particular, the disclosure relates to a method for reducing and / or preventing weight gain in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei. The at least one strain of Lacticaseibacillus paracasei is suitable for use in supporting and / or prompting and / or maintaining weight control in a subject.

[0032] Thus, an object of the present invention is to provide a method for reducing and / or preventing weight gain in a subject in need thereof.

[0033] Therefore, a first aspect of the invention provides a method for reducing and / or preventing weight gain in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0034] A second aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in reducing and / or preventing weight gain in a subject in need thereof.

[0035] A third aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

[0036] A fourth aspect of the invention provides a non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

[0037] A fifth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in reducing and / or preventing weight gain in a subject in need thereof.

[0038] A sixth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a supplement for use in reducing and / or preventing weight gain in a subject in need thereof. A seventh aspect of the invention provides a method for supporting and / or promoting and / or maintaining weight control in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0039] An eighth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use supporting and / or promoting and / or maintaining weight control in a subject.

[0040] A ninth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for supporting and / or promoting and / or maintaining weight control in a subject.

[0041] A tenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in supporting and / or promoting and / or maintaining weight control in a subject.

[0042] An eleventh aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for supporting and / or promoting and / or maintaining weight control in a subject.

[0043] A twelfth aspect of the invention provides a method for maintaining and / or supporting a healthy body weight in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei, optionally wherein the healthy body weight is a healthy body mass index (BMI).

[0044] A thirteenth aspect of the invention provides the at least one strain of Lacticaseibacillus paracasei for use in maintaining and / or supporting a healthy body weight in a subject.

[0045] A fourteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting a healthy body weight in a subject.

[0046] A fifteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining and / or supporting a healthy body weight in a subject.

[0047] A sixteenth aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting a healthy body weight in a subject. A seventeenth aspect of the invention provides the a method for maintaining and / or supporting healthy body mass index (BMI) in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0048] An eighteenth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0049] A nineteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0050] A twentieth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0051] A twenty-first aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0052] A twenty-second aspect of the invention provides a method for the prevention and / or amelioration and / or treatment of metabolic syndrome in a subject, comprising administering an effective dose of at least one strain of Lacticaseibacillus paracasei to a subject in need thereof.

[0053] A twenty-third aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

[0054] A twenty-fourth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for the manufacture of a medicament for use in preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

[0055] A twenty-fifth aspect of the invention provides a method for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0056] A twenty-sixth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

[0057] A twenty-seventh aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

[0058] A twenty-eighth aspect of the invention provides a method for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0059] A twenty-ninth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

[0060] A thirtieth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

[0061] A thirty-first aspect of the invention provides a method for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0062] A thirty-second aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof.

[0063] A thirty-third aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof. A thirty-fourth aspect of the invention provides a method for maintaining normal blood cholesterol levels in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0064] A thirty-fifth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining normal blood cholesterol levels in a subject.

[0065] A thirty-sixth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining normal blood cholesterol levels in a subject.

[0066] A thirty-seventh aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining normal blood cholesterol levels in a subject.

[0067] A thirty-eighth aspect of the invention provides a method for supporting and / or maintaining endothelial function in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0068] A thirty-ninth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining endothelial function in a subject.

[0069] A fortieth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining endothelial function in a subject.

[0070] A forty-first aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining endothelial function in a subject.

[0071] A forty-second aspect of the invention provides a method for maintaining healthy blood lipid and / or blood fat in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0072] A forty-third aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining healthy blood lipid and / or blood fat in a subject. A forty-fourth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining healthy blood lipid and / or blood fat in a subject.

[0073] A forty-fifth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining healthy blood lipid and / or blood fat in a subject.

[0074] A forty-sixth aspect of the invention provides a method for aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0075] A forty-seventh aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0076] A forty-eighth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0077] A forty-ninth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0078] The present invention will in the following be described in more detail.

[0079] BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1. Summary of study design. (A) Schematic diagram of the study design. (B) Flowchart of enrolment and randomization. + 50 participants (91%) in the L. paracasei group and 46 participants (95%) in the placebo group completed all 3 visits. ITT = intention-to-treat. L. paracasei = Lacticaseibacillus paracasei 8700:2.

[0081] Figure 2. Ameliorative effect of L. paracasei on endothelial function. Adjusted means of change in FMS and two-sided 95%CIs for each group and the pairwise comparisons between two groups, from baseline to week 12. Subgroup indicates participants with more severe endothelial dysfunction defined as baseline FMS < 17%. FMS = flow-mediated slowing. ITT = intention-to-treat. L. paracasei = Lacticaseibacillus paracasei 8700:2. PP = per-protocol.

[0082] Figures 3. Beneficial effect of L. paracasei on lipid profiles and its association with improved endothelial function. (A, B) Intention-to-treat analysis and (C, D) per- protocol analysis for adjusted mean change in (A, C) TG and (B, D) remnant cholesterol and two-sided 95%CIs for each group and the pairwise comparisons between two groups, from baseline to week 12, respectively. Correlations between reduction in remnant cholesterol and improvement in FMS from baseline to week 12 in (E) intention-to-treat and (F) per-protocol analysis, controlling for age, gender and baseline values. Due to missing values, the numbers of pairs used in (E) and (F) were 100 and 87, respectively. Correlation coefficients and P values derived from Partial Correlations Analysis were presented. FMS = flow-mediated slowing. ITT = intention-to-treat. L. paracasei Vi Lacticaseibacillus paracasei 8700:2. PP = per- protocol. TG = triglyceride.

[0083] Figure 4. Effect of L. paracasei on TC, HDL-c and LDL-c. (A-C) Intention-to-treat analysis and (D-F) per-protocol analysis for adjusted mean change in (A, D) TC, (B, E) HDL-c, and (C, F) LDL-c and two-sided 95%CIs for each group and the pairwise comparisons between two groups, from baseline to week 12, respectively. ITT = intention-to-treat. TC = total cholesterol. HDL-c = high-density lipoprotein cholesterol. LDL-c = low-density lipoprotein cholesterol. L. paracasei = Lacticaseibacillus paracasei 8700:2. PP = per-protocol.

[0084] Figure 5. Alleviative effect of L. paracasei on the severity of metabolic syndrome. (A, B, E, F) Intention-to-treat analysis and (C, D, G, H) per-protocol analysis for adjusted mean change in (A, C) number of MetS components, (B, D) MetS severity, (E, G) body weight, and (F, H) body mass index and two-sided 95%CIs for each group and the pairwise comparisons between two groups, from baseline to week 12, respectively. ITT = intention-to-treat. L. paracasei = Lactobacillus paracasei 8700:2. MetS = metabolic syndrome. PP = per-protocol. DETAILED DESCRIPTION

[0085] The disclosure relates to a method for reducing and / or preventing weight gain in a subject. In particular, the disclosure relates to a method for reducing and / or preventing weight gain in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei. The at least one strain of Lacticaseibacillus paracasei is suitable for use in supporting and / or prompting and / or maintaining weight control in a subject.

[0086] The inventors of the present invention have surprisingly succeeded in developing a method for reducing and / or preventing weight gain in a subject. It has been successfully demonstrated in the experimental examples, that administration of probiotic bacteria strain Lacticaseibacillus paracasei according to the invention has surprisingly led to the positive effect of reducing weight gain, decreasing BMI, and increasing endothelial function in subjects.

[0087] Thus, a first aspect of the invention provides a method for reducing and / or preventing weight gain in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0088] By "weight gain", we include an increase in total body weight in a subject. Weight gain can involve an increase in muscle mass, fat deposits, excess fluid such as water or other factors. Preferably, the weight gain is the result of an increase in the fat deposits in a subject and / or is primarily due to increase in fat deposits in a subject. Weight gain may be measured on a scale or on a body composition monitor.

[0089] By "reducing and / or preventing" we include the meaning of a use which gives rise to an effect in a subject of preventing, protecting against, reducing the severity of, removing, blocked from occurring or made to cease. Prevention typically takes place before the event occurs or the effect or condition is manifest, but it will be appreciated that it can also mean to prevent further occurrence of the same kind of event. It will also be appreciated that such terms may include the meaning that an event or condition is maintained in the current state without becoming worse or developing further. Reducing typically takes place when the event or condition has already occurred and it means to make something smaller or less in amount, degree or size. The reduction may be relative to the level of the given parameter (e.g. weight gain) had the subject not taken the at least one strain of L. paracasei. Alternatively, the reduction may be relative to the level of the parameter (e.g. weight gain) before the supplementation period and / or prior to receiving the at least one strain of L. paracasei, uses thereof or methods of the invention.

[0090] A second aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in reducing and / or preventing weight gain in a subject in need thereof.

[0091] A third aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

[0092] A fourth aspect of the invention provides a non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

[0093] By "non-therapeutic use" we include the meaning of non-medical use, which is to be understood as not relating to, being, or providing therapy. The terms is to be understood as covering cosmetic uses and methods. In particular, the term covers cosmetic weight reduction, optionally where no direct health-related benefits can be derived from the weight reduction. The present invention covers instances of preventing weight gain for cosmetic reasons.

[0094] A fifth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in reducing and / or preventing weight gain in a subject in need thereof.

[0095] A sixth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a supplement for use in reducing and / or preventing weight gain in a subject in need thereof.

[0096] A seventh aspect of the invention provides a method for supporting and / or promoting and / or maintaining weight control in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0097] By "weight control" we include the meaning of remain managing body weight, and preferably managing body weight to remain weight neutral or to prevent weight gain. Weight control also refers to weight management which is the practice of behaviours, techniques and physiological processes that contributes to an individual's ability to maintain a healthy weight and / or prevent weight gain. An eighth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use supporting and / or promoting and / or maintaining weight control in a subject.

[0098] By "supporting and / or promoting and / or maintaining" we include the meaning of enabling weight control by helping or aiding a subject to remain weight neutral or preventing weight gain.

[0099] A ninth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for supporting and / or promoting and / or maintaining weight control in a subject.

[0100] A tenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in supporting and / or promoting and / or maintaining weight control in a subject.

[0101] An eleventh aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for supporting and / or promoting and / or maintaining weight control in a subject.

[0102] A twelfth aspect of the invention provides a method for maintaining and / or supporting a healthy body weight in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei, optionally wherein the healthy body weight is a healthy body mass index (BMI).

[0103] A thirteenth aspect of the invention provides the at least one strain of Lacticaseibacillus paracasei for use in maintaining and / or supporting a healthy body weight in a subject.

[0104] By "body weight" we include the subject's mass or weight. Body weight is typically expressed in kilograms or pounds. Body weight can be used to calculate a subject's body mass index (BMI).

[0105] By "body mass index (BMI)", we include a method of estimating the total body fat of a subject. BMI is calculated by an individual's weight in kilograms divided by the square of height in meters.

[0106] BMI can be used as a medical screening tool to categorise the degree of body fatness and obesity, but it cannot necessarily be used to diagnose the body fatness or health of an individual. A high BMI may indicate high body fatness. A subject with a BMI of less than 18.5 is considered to be underweight. A subject with a BMI of 18.5 to 24.9 is considered to be healthy (or to have a healthy body weight - see below). A subject with a BMI from 25.0 to 29.9 is considered to be overweight. A subject is considered obese when the BMI level is at 30 or higher.

[0107] By "healthy body weight" we include the meaning of an individual's body weight that is associated with low risk of weight-related diseases and health issues. A healthy body weight also refers to normal or ideal body weight which should be understood as an appropriate weight in relation to height or the optimal weight associated with the maximum life expectancy for a given height. Alternatively, a healthy body weight may also be defined as a body mass index (BMI) within the range of 18.5 to 24.9.

[0108] A fourteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting a healthy body weight in a subject.

[0109] A fifteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining and / or supporting a healthy body weight in a subject.

[0110] A sixteenth aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting a healthy body weight in a subject.

[0111] A seventeenth aspect of the invention provides the a method for maintaining and / or supporting healthy body mass index (BMI) in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0112] By "healthy body mass index (BMI)", we include the meaning of a BMI within the range of 18.5 to 24.9.

[0113] Maintaining a healthy body weight and healthy BMI has a number of health benefits such as decreasing the risk of developing high blood pressure, diabetes, heart disease, and stroke as well as lowering the risk of several types of cancer. Decreasing the risk of these diseases will inevitably lead to increase quality of life and a longer lifespan. Maintaining a healthy body weight and healthy BMI involves making long-term lifestyle changes, which includes healthy eating, balancing the number of calories eaten with the number of calories burned or regulated by physical exercise.

[0114] Dietary supplementation of probiotic strains such as Lacticaseibacillus paracasei has been demonstrated to effectively slow down body weight gain and reduce fat accumulation in mice exposed to a Western diet. Hence, it may therefore be advantageous to provide at least one strain of Lacticaseibacillus paracasei in a method of maintaining and / or supporting healthy BMI in a subject.

[0115] Thus, an eighteenth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0116] A nineteenth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0117] A twentieth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0118] A twenty-first aspect of the invention provides the non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for maintaining and / or supporting healthy body mass index (BMI) in a subject.

[0119] The disclosure also relates to methods for preventing, ameliorating or treating metabolic syndrome (MetS) in a subject and / or improving card io meta bo lie health and / or cardiovascular health using an effective amount of at least one strain of Lacticaseibacillus paracasei. The disclosure further relates to methods for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease. The disclosure further relates to methods for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject.

[0120] It has been successfully demonstrated in the experimental examples, that administration of probiotic bacteria strain Lacticaseibacillus paracasei has various cardiometabolic benefits. This includes, but is not limited to, reduced remnant cholesterol, enhanced endothelial function, lower triglycerides, alleviation of MetS severity. Therefore, L. paracasei can be utilised in a range of methods, including preventing, ameliorating or treating metabolic syndrome (MetS); improving cardiometabolic and / or cardiovascular health; reducing risk factors associated with cardiometabolic diseases; reducing risk factors associated with cardiovascular diseases; preventing and / or ameliorating cardiometabolic disease; preventing and / or ameliorating cardiometabolic disease cardiovascular disease; maintaining normal blood cholesterol; supporting and / or maintaining endothelia function; maintaining healthy blood lipid and / or blood fat; and / or aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining.

[0121] By "Metabolic syndrome" we include the meaning of a physical condition that refers to several risk factors specific for cardiovascular disease. Metabolic syndrome covers five metabolic abnormalities, which includes (i) obesity, (ii) high blood pressure, (iii) high blood triglycerides, (iv) low levels of HDL cholesterol and (v) insulin resistance or impaired fasting blood glucose.

[0122] The obesity is preferably abdominal obesity, which includes having a waist circumference of more than 35 inches for women and more than 40 inches for men. An increased waist circumference is the form of obesity most strongly tied to metabolic syndrome.

[0123] By "high pressure", we include a blood pressure of 130 / 80 mm Hg (millimetres of mercury) higher. Normal blood pressure is defined as less than 120 mm Hg for systolic pressure (the top number), and less than 80 mm Hg for diastolic pressure (the bottom number). High blood pressure is strongly tied to obesity. It is often found in people with insulin resistance.

[0124] By "impaired fasting blood glucose", we include a fasting blood glucose level equal to or greater than 100 mg / dL.

[0125] By "high triglyceride levels" we include a triglyceride level of more than 150 mg / dL. Triglycerides are a type of fat in the blood.

[0126] By "low" levels of HDL (good) cholesterol, we include a HDL level of less than 40 mg / dL for men and less than 50 mg / dL for women.

[0127] Metabolic syndrome is a condition that greatly raises the risk of developing type II diabetes, heart disease and stroke. The risk factors for developing metabolic syndrome are family history of premature coronary disease, hypertension, hyperlipemia, diabetes and smoking. Age increases the risk of metabolic syndrome, as does male gender and post-menopausal hormonal status. Certain risk factors tend to cluster or occur together and some of these risk factors can be deceased by lifestyle modifications of dietary changes and increased physical activity combined with pharmacologic treatment, which may affect several risk factors simultaneously and thereby reduce the risk of developing metabolic syndrome.

[0128] Modulating the gut microbiota to decrease some of the risk factors of developing metabolic syndromes may be an attractive strategy, as it has been demonstrated in both mice and human studies that there is a close link between changes in gut microbiota and the development of metabolic syndrome.

[0129] Therefore, it may be advantageous to provide at least one strain of Lacticaseibacillus paracasei in a method for preventing and / or ameliorating and / or treating of metabolic syndrome in a subject.

[0130] Thus, a twenty-second aspect of the invention provides a method for the prevention and / or amelioration and / or treatment of metabolic syndrome in a subject, comprising administering an effective dose of at least one strain of Lacticaseibacillus paracasei to a subject in need thereof.

[0131] A twenty-third aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

[0132] A twenty-fourth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei for the manufacture of a medicament for use in preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

[0133] By "treating" we include the meaning of an event or condition being treated. Treating includes ameliorating, reducing in severity, removing, blocking from occurring further, protecting against occurring further, delaying and / or ceasing. Such treatment typically takes place after the event (or the same kind of event) has occurred or the condition manifests. It will also be appreciated that such terms may include the meaning that an event or condition is maintained in the current state without becoming worse or developing further. By "ameliorating" we include the meaning of an event or condition being ameliorated. Ameliorating is improving, enhancing, reduced in severity, and / or making tolerable or acceptable. Such ameliorating typically takes place after the event (or the same kind of event) has occurred or the condition is manifest.

[0134] In one embodiment, the metabolic syndrome comprises one or more of the following parameters: a) reduced endothelial function; and / or b) increased body weight gain; and / or c) increased body mass index (BMI); and / or d) increased waist circumference; and / or e) increased blood pressure; and / or f) increased triglyceride levels; and / or g) increased total cholesterol; and / or h) increased remnant cholesterol; and / or i) increased low-density lipoprotein cholesterol (LDL); and / or j) decreased high-density lipoprotein cholesterol (HDL-c); and / or k) increased metabolic severity (e.g. increased metabolic severity score (MetS-Z); and / or l) reduced and / or impaired glucose metabolism; and / or m) increased fasting blood glucose; and / or n) insulin resistance or reduced insulin sensitivity.

[0135] By "endothelial function", we include the meaning of the function the endothelium, which is a major organ consisting of a single cell layer of endothelial cells that forms the lining of the vascular an lymphatic system in mammals. The endothelial function may be measured by any suitable method known in the art, including non-invasive methods and invasive methods.

[0136] Noninvasive methods of measuring endothelial function include ultrasound FMD, salbutamol-mediated endothelial function measured by pulse wave analysis (PWA) or pulse contour analysis (PCA), flow-mediated magnetic resonance imaging (MRI), laser Doppler flowmetry, and flow-mediated pulse amplitude tonometry (PAT). Noninvasive ultrasound FMD of the brachial artery is the most widely used method for both small and large population studies of adults and children Invasive methods for measuring endothelial function include intra-arterial ACh or edothelin infusion and strain gauge plethysmography (or high resolution ultrasound) and intravascular studies of epicardial coronary arteries.

[0137] Preferably endothelial function is measured by ultrasound using flow-mediated dilation (FMD) of the brachial artery and by measuring the concentration of triglycerides in peripheral blood.

[0138] A reduced endothelial function is to be understood as endothelial dysfunction which may increases the risk of health issues such as heart disease and atherosclerosis. A reduced endothelial function is considered to be less than around 6.5% ±0%? Normal endothelial function measured by FMD is considered to be in the range of 6.5 - 10%. Typically there is a significant age-related decline in endothelial function.

[0139] By "body weight gain" or "weight gain", we include an increase in total body weight in a subject. Weight gain can involve an increase in muscle mass, fat deposits, excess fluid such as water or other factors. Preferably, the weight gain is the result of an increase in the fat deposits in a subject or is primarily due to increase in fat deposits in a subject. Weight gain may be measured on a scale or on a body composition monitor.

[0140] By increased body weight gain, we mean above a normal or ideal body weight. This is dependent on the height, age and gender of the individual. For example, it could mean that the subject has a BMI that is higher than the normal range.

[0141] By "waist circumference" we include the meaning of a measure of body fat distribution by measuring the distance around the waist. The waist circumference may be used as an health indicator, as increased waist circumference may be linked to a higher risk of various health conditions such as metabolic syndrome. The waist circumference may be measured using a tape measure. An increased waist circumference may be an increase relative to a healthy subject. An increased waist circumference may be considered to be greater than 94 cm for a male subject and / or greater than 80 cm for a female subject. A normal waist circumference is considered to be less than or equal to 94 cm for a male subject and less than or equal to 80 cm for a female subject.

[0142] Waist circumference is also referred to as waistline circumference, and the terms may be used interchangeably. By "blood pressure" we include the meaning of the pressure, measured in millimetres of mercury, within the major arterial system of the body. It is separated into systolic and diastolic pressure, where systolic pressure is the maximum blood pressure during contraction of the ventricle and diastolic pressure is the minimum blood pressure measured just prior to the next contraction. The blood pressure is written as the systolic pressure over the diastolic pressure, e.g. 120 / 80 mm Hg (millimetres of mercury).

[0143] Blood pressure may be measured by automatic electronic sphygmomanometer after resting for more than 5 minutes. Increased blood pressure is considered to be a blood pressure of 130 / 80 mm Hg or higher and a normal blood pressure is considered to be a blood pressure in the range of 90 / 60 mm Hg to 120 / 80 mm Hg, typically with slightly higher values with increased age.

[0144] By "triglyceride levels" we include the meaning of the level of lipid or fat in the blood plasma. Increased triglyceride levels can raise the risk of heart attack and stroke. Triglyceride levels may be measured by blood sampling of peripheral blood and analysed by nuclear magnetic resonance (NMR) spectroscopy.

[0145] Normal triglyceride levels are gender-specific. A normal triglyceride level for a male subject will be in the range 40-160 mg / dL and for a female subject will be in the range 35-135 mg / dL. By "increased triglyceride level", we include a triglyceride level that is higher than the normal range for a given subject. For example, for female subjects an increased triglyceride level is considered to be above 135 mg / dL, preferably in the range of 135-500 mg / dL. For female subjects an increased triglyceride level is considered to be above 160 mg / dL, preferably in the range of 160-500 mg / dL.

[0146] By "total cholesterol" we include the meaning of the total amount of cholesterol in the blood plasma. It includes both low-density lipoprotein (LDL) cholesterol, high-density lipoprotein cholesterol (HDL) and 20% of the body's triglycerides. Total cholesterol may be measured by blood sampling of peripheral blood and analysed by nuclear magnetic resonance (NMR) spectroscopy. An increased total cholesterol level is considered to be 200 mg / dl or higher and a normal total cholesterol level is considered to be less than 200 mg / dL.

[0147] By "remnant cholesterol" we include the meaning of the amount of cholesterol in a form of very low-density lipoprotein (VLDL) in the blood plasma. Increased levels of remnant cholesterol in the blood may form plaque in the arteries and lead to atherosclerosis. Remnant cholesterol may be measured by taking the value of total cholesterol and minus the HDL and LDL values. Further, remnant cholesterol may be measured by blood sampling of peripheral blood and analysed by nuclear magnetic resonance (NMR) spectroscopy. An increased remnant cholesterol level is considered to be 0.77 mmol / L or higher or more than 30 mg / dL and a normal remnant cholesterol level is considered to be in the range of 0.01 to 0.77 mmol / L or below 30 mg / dL.

[0148] By "low-density lipoprotein cholesterol (LDL)" we include the meaning of lipid or fat that circulates in the blood. LDL has a role in transporting cholesterol around the body and is commonly refers to as "bad" cholesterol, as increased LDL levels can lead to plaque formation in the arteries, causing atherosclerosis. LDL may be measured by blood sampling of peripheral blood and analysed by nuclear magnetic resonance (NMR) spectroscopy. An increased LDL level is considered to 100 mg / dL and a normal LDL level is considered to be below 100 mg / dL.

[0149] By "high-density lipoprotein cholesterol (HDL-c)" we include the meaning of lipid or fat that circulates in the blood. HDL-c has a role in transporting cholesterol from parts of the body back to the liver where it gets removed from the body. HDL-c is commonly referred to as "good" cholesterol, as it removes other forms of cholesterol form the bloodstream. Decreased level of HDL-c is associated with metabolic syndrome. HDL-c may be measured by blood sampling of peripheral blood and analysed by nuclear magnetic resonance (NMR) spectroscopy. A decreased HDL-c level is considered to below 40 mg / dL and a normal HDL-c level is considered to be between 40-60 mg / dL, with HDL-c levels of above 60 ml / dL an increased HDL-c level (i.e. even better).

[0150] High-density lipoprotein cholesterol (HDL-c) also refers to as HDL, and the terms may be used interchangeably.

[0151] By "metabolic severity" we include the meaning of a metabolic syndrome severity Z score (MetS-Z), which is a clinical prediction rule for diagnostic and therapeutic decisions and uses the following parameters to predict the score: gender, age, race, systolic blood pressure, waistline circumference, high-density lipoprotein (HDL), triglycerides and fasting blood glucose. The Mets-Z indicates the current severity status of metabolic syndrome in an individual compared to others in a given population. A MetS-Z score of 0 for a given individual indicates the value for that individual is equal to the mean of the population. A score below 0 means that the individual has lower than average risk, while a score above 0 means the individual has higher than average metabolic syndrome severity.

[0152] By "glucose metabolism" we include the meaning of a biochemical process by which sugar molecules from the food are processed and used to produce energy in the form of ATP. Glucose metabolism may be measured by oral glucose tolerance test, where 75g glucose is administered orally following blood sampling 2 hours post challenge and measurement of glucose level in the blood plasma. Blood samples may be collect at time point 0, 60 and 120 minutes. The glucose level in the blood plasma may be measured by enzymatic methods, such as hexokinase, glucose oxidase and glucose dehydrogenase.

[0153] Reduced and / or impaired glucose metabolism means that the blood glucose is raised beyond normal level, but not high enough to warrant a diabetes diagnosis and it is defined as a 2-hour post challenge glucose level between 140 and 200 mg / dL during oral glucose tolerance test. A normal glucose metabolism is a blood glucose level between 120 to 140 mg / L during oral glucose tolerance test, typically with slightly higher values with increased age.

[0154] By "fasting blood glucose" we include the meaning of the level of glucose in the blood after an individual has been fasting, or not consumed any food or beverage, except for water, for at least eight hours.

[0155] Fasting blood glucose may be measured by blood sampling of peripheral blood after at least eight hours of fasting. The glucose level in the blood plasma may be measured by enzymatic methods, such as hexokinase, glucose oxidase and glucose dehydrogenase.

[0156] Impaired fasting blood glucose is considered to be equal or greater than lOOmg / dL and a normal fasting blood glucose is considers to be below 100 mg / dL. Individuals whose blood glucose is in the range of 100 md / dL to 125 mg / dL can be diagnosed with prediabetes and individuals whose blood glucose is at or above 126 mg / dL can be diagnosed with diabetes.

[0157] By "insulin resistance or reduced insulin sensitivity" we include the meaning of the cells responsiveness to insulin. Insulin resistance or reduced insulin sensitivity is also to be understood as diabetes. Insulin is a hormone that help the body control the level of glucose in the blood. Individuals with insulin resistance or reduced insulin sensitivity has built up a tolerance to insulin, making the hormone less effective. As a result, more insulin is needed for the fat cells, muscle cells and the liver to respond to the insulin and take up glucose from the blood to store it.

[0158] Insulin resistance or reduced insulin sensitivity may be measure by haemoglobin Ale test, insulin test, hyperinsulinemic euglycemic clamp and / or intravenous glucose tolerance test. The haemoglobin Ale level may be determined by high performance liquid chromatography and the inulin level may be determined by an enzyme-linked immunosorbent assay.

[0159] A normal haemoglobin Ale level in the blood plasma is considered to be in the range of 4% to 5.6%. A haemoglobin Ale level within the range of 5.7% to 6.4% is considered to be prediabetic. Insulin resistance or reduced insulin sensitivity is diagnosed if the haemoglobin Ale level is of 6.5% or above.

[0160] A twenty-fifth aspect of the invention provides a method for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0161] By "cardiometabolic health and / or cardiovascular health" we include the meaning of the health of an individual's cardiovascular system, including heart, blood and blood vessels.

[0162] Cardiometabolic disease covers a group of common but often preventable conditions including heart attack, stroke, diabetes, insulin resistance, non-alcoholic fatty liver disease. These is a global increase in the number of people who experience one or more of these conditions during their lifetime. Prevention of cardiometabolic disease is through lifestyle changes of eating and drinking habits, physical exercise or by pharmacotherapy.

[0163] Cardiovascular disease covers a group of disorders of the heart and blood vessels and includes coronary heart disease, cerebrovascular disease, rhematic heart disease, stroke, peripheral arterial disease and aortic disease. Cardiovascular disease are usually associated with build-up of fatty deposits inside the arteries, also referred to as atherosclerosis and an increased risk of blood clots. It may also be associated with damage to arteries in organs such as the brain, heart, kidney and eyes. Cardiovascular disease is one of the main causes of deaths and disabilities globally and can often be prevented by committing to a heathier lifestyle. However, since incidences of both cardiometabolic disease and cardiovascular disease are increasing each year, there is a need for new or alternative method for preventing and / or treating cardiometabolic disease and cardiovascular disease.

[0164] Preclinical models and human studies have demonstrated a close link between change in gut microbiota and the development of cardiometabolic disease and cardiovascular disease through several mechanisms, such as increased production of lipopolysaccharide, disturbance in gut immune signalling, and perturbation in the metabolism of bile acid, short-chain fatty acids, trimethylamine-N-oxide, and uremic toxin. Therefore, modulating the gut microbiota may be an attractive strategy for treating and / or preventing cardiometabolic disease and cardiovascular disease by improving cardiometabolic health and cardiovascular health through administration of probiotic strains as a diets supplement. Especially utilizing probiotic strains belonging to Lactobacillus and Bifidobacteria species would be an attractive approach, as several strains has demonstrated a favourable effect on host metabolism, including Lacticaseibacillus paracasei.

[0165] Therefore, a twenty-sixth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

[0166] A twenty-seventh aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

[0167] A twenty-eighth aspect of the invention provides a method for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0168] A twenty-ninth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

[0169] A thirtieth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

[0170] A thirty-first aspect of the invention provides a method for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei. A thirty-second aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof.

[0171] A thirty-third aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof.

[0172] A thirty-fourth aspect of the invention provides a method for maintaining normal blood cholesterol levels in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0173] By "normal blood cholesterol levels" we include the meaning of total cholesterol measured in blood plasma to be less than 200 mg / dL.

[0174] A thirty-fifth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining normal blood cholesterol levels in a subject.

[0175] A thirty-sixth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining normal blood cholesterol levels in a subject.

[0176] A thirty-seventh aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining normal blood cholesterol levels in a subject.

[0177] In one embodiment, the subject already has a blood cholesterol level within the normal range.

[0178] The endothelial function has a significant role in various physiological functions including controlling blood fluidity, vascular tone, permeability, anticoagulation and angiogenesis. The endothelium is a large organ that plays a key role in keeping the blood moving smoothly through the body and it is made up of over a trillion endothelial cells, which releases substances that aids the blood flow. Endothelial dysfunction is of significance in predicting stroke and heart attack due to the inability of the arteries to dilate fully. A reduced endothelial function may occur as a result of high blood pressure, diabetes, high cholesterol and smoking. Thus, it may be advantageous to support and / or maintain endothelial function in a subject by administering at least one strain of Lacticaseibacillus paracasei.

[0179] Therefore, a thirty-eighth aspect of the invention provides a method for supporting and / or maintaining endothelial function in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0180] A thirty-ninth aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining endothelial function in a subject.

[0181] A fortieth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining endothelial function in a subject.

[0182] A forty-first aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining endothelial function in a subject.

[0183] A forty-second aspect of the invention provides a method for maintaining healthy blood lipid and / or blood fat in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0184] By "healthy blood lipid and / or blood fat" we include the meaning of cholesterol and triglycerides in the blood within normal levels, which is considered to be less than 200 mg / dL for cholesterol and less than 150 mg / dL for triglycerides.

[0185] A forty-third aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in maintaining healthy blood lipid and / or blood fat in a subject.

[0186] A forty-fourth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for maintaining healthy blood lipid and / or blood fat in a subject.

[0187] A forty-fifth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in maintaining healthy blood lipid and / or blood fat in a subject.

[0188] By "aiding and / or assisting repair" we include the meaning of help to restore damaged tissue in an organ or help to return to a normal status and / or healthy status of the tissue. By "gastrointestinal wall lining and / or gut wall lining" we include the meaning of the four layers of tissue in the gastrointestinal wall lining and / or gut wall lining. From the inner cavity of the gut there are the mucosa, the submucosa, the muscular layer and the serosa. The mucosa is the innermost layer of the gastrointestinal tract.

[0189] A forty-sixth aspect of the invention provides a method for aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

[0190] A forty-seventh aspect of the invention provides at least one strain of Lacticaseibacillus paracasei for use in aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0191] A forty-eighth aspect of the invention provides use of at least one strain of Lacticaseibacillus paracasei for aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0192] A forty-ninth aspect of the invention provides the use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament or supplement for use in aiding and / or assisting repair of gastrointestinal wall lining and / or gut wall lining in a subject.

[0193] In one embodiment the present invention relates to the method, the at least one strain or the use as described herein, wherein the subject has metabolic syndrome.

[0194] In one embodiment, the present invention relates to the method, the at least one strain or the use as described herein, wherein the subject has metabolic syndrome or increased risk for any of the symptoms included in the metabolic syndrome or dysregulated metabolic health.

[0195] Another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the subject is a mammal, preferably wherein the subject is human.

[0196] In any of the aspects or embodiments described herein, the subject has an increased BMI. In any of the aspects or embodiments described herein, the subject has recovered from increased BMI and / or metabolic syndrome. In any of the aspects or embodiments described herein, the subject has recovered from increased BMI and / or metabolic syndrome recovered from increased BMI and / or metabolic syndrome and wants to maintain their weight loss and / or health improvement. In any of the aspects or embodiments described herein, the subject has recovered from cardiometabolic disease. In any of the aspects or embodiments described herein, the subject has recovered from cardiometabolic disease and wants to maintain their weight loss and / or health improvement. In any of the aspects or embodiments described herein, the subject has recovered from a cardiovascular disease. In any of the aspects or embodiments described herein, the subject has recovered from a cardiovascular disease and wants to maintain their weight loss and / or health improvement.

[0197] The subject may belong to any or all of the aforementioned groups, e.g. at least one, two, three, four, five etc of the aforementioned groups.

[0198] The at least one Lacticaseibacillus / Lactobacillus strain according to the present invention may be viable, inactivated or dead. Preferably, the strain is viable. For example, preferably the strain is freeze-dried.

[0199] Advantageously, the at least one strain of Lacticaseibacillus or Lactiplantibacillus is a probiotic strain.

[0200] Probiotic bacteria are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Hill et a / , Nat Rev Gastroenterol Hepatol, 2014, 11(8): 506-514). For a bacterium to fulfil the definition of a probiotic it typically has to be able to survive in and colonise the intestines, survive the processes of production and storage, and have evidence that it has positive effects on consumer health.

[0201] By "probiotic strain" we include the meaning of a strain of bacteria which when 5 administered in adequate amounts confer a health benefit on the host. Typically, the administration of said at least one probiotic strain will alter the composition of the gut microbiota.

[0202] By "at least one strain" we include the meaning of one or more strain(s) of bacteria which when administered in adequate amounts confer a health benefit on the host. Preferably the "at least one strain" according to the present invention is at least one strain of Lacticaseibacillus paracasei DSM 13434. The Lacticaseibacillus paracasei species was formerly known by the name Lactobacillus paracasei. The terms "Lacticaseibacillus paracasei", "L. paracasei" and "Lactobacillus paracasei" are used interchangeably and refers to the same.

[0203] The Lactiplantibacillus plantarum species was formerly known by the name Lactobacillus plantarum. The terms "Lactiplantibacillus plantarum", "L. plantarum" and "Lactobacillus plantarum" are used interchangeably and refers to the same.

[0204] Thus, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain of Lacticaseibacillus paracasei comprises Lacticaseibacillus paracasei DSM 13434 (8700:2™) or Lacticaseibacillus paracasei DSM 13432 (02A™). Preferably, wherein the at least one strain of Lacticaseibacillus paracasei is Lacticaseibacillus paracasei DSM 13434 (8700:2™).

[0205] A further embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain is Lacticaseibacillus paracasei DSM 13434 (8700:2™) in combination with at least one strain of Lactiplantibacillus plantarum.

[0206] Yet, a further embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain of Lactiplantibacillus plantarum is selected from Lactiplantibacillus plantarum DSM 6595 (299™), Lactiplantibacillus plantarum DSM 9843 (299v®), Lactiplantibacillus plantarum DSM 15312 (HEAL 9), Lactiplantibacillus plantarum DSM 15313 (HEAL 19™), Lactiplantibacillus plantarum DSM 15316 (HEAL 99™), Lactiplantibacillus plantarum DSM 32131 (GOS42™), Lactiplantibacillus plantarum DSM 17852 (LB3e™) and Lactiplantibacillus plantarum DSM 17853 (LB7c™). Preferably, wherein the at least one strain is Lacticaseibacillus paracasei DSM 13434 (8700:2™) in combination with Lactiplantibacillus plantarum DSM 9843 (299v®).

[0207] In an embodiment according to the invention, the at least one strain is present (e.g. in a composition) in an amount from about IxlO6to about IxlO14CFU / dose, preferably from about IxlO8to about IxlO12CFU / dose, more preferably from about IxlO9to about IxlO11CFU / dose, and most preferably about IxlO10CFU / dose. If the at least one strain consists of more than one strain, such amounts represent the total CFU / dose of the combination of strains. For example, the at least one strain may be present in an amount from about IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12or about IxlO13CFU / dose. The at least one strain may be present in an amount to about IxlO14, IxlO13, IxlO12, IxlO11, IxlO10, IxlO9, IxlO8or about IxlO7CFU / dose. The at least one strain according to the invention may also be used alone in water or any other aqueous vehicle in which the at least one strain is added or mixed before ingestion.

[0208] In an embodiment, the at least one strain of Lacticaseibacillus paracasei is present in an amount from about IxlO9to about 10xl09CFU, such as IxlO9CFU. In an embodiment, the at least one strain of Lactiplantibacillus plantarum is present in an amount from about IxlO9to about 10xl09CFU, such as IxlO9CFU.

[0209] In an embodiment, at least one strain of Lacticaseibacillus paracasei is present in an amount from about IxlO9to about 10xl09CFU and the at least one strain of Lactiplantibacillus plantarum is present in an amount from about IxlO9to 10xl09about CFU.

[0210] In an embodiment, the at least one strain is Lacticaseibacillus paracasei DSM 13434 (8700:2™) in combination with Lactiplantibacillus plantarum DSM 9843 (299v®) and the Lacticaseibacillus paracasei DSM 13434 (8700:2™) is present in an amount from about IxlO9to about 10xl09CFU (preferably IxlO9CFU) and the Lactiplantibacillus plantarum DSM 9843 (299v®) is present in an amount from about IxlO9to about 10xl09CFU (preferably IxlO9). In any embodiment, the CFU may be the CFU per dose.

[0211] Lactobacillus paracasei DSM 13434 (8700:2™) and Lactobacillus paracasei DSM 13432 (02:A™) were deposited on 6 April 2000 at DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, by Probi AB.

[0212] Lactobacillus plantarum DSM 6595 (299™) was deposited on 2 July 1991 at DSM- DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg 1 B, D-3300 Braunschweig, Germany, in the name of Probi (i.e. Probi AB).

[0213] Lactobacillus plantarum DSM 9843 (299V®) was deposited on 16 March 1995 at DSM- DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, by Probi AB.

[0214] Lactobacillus plantarum DSM 15312 (HEAL 9™), Lactobacillus plantarum DSM 15313 (HEAL 19™), and Lactobacillus plantarum DSM 15316 (HEAL 99™), were deposited on 27 November 2002 at DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, by Probi AB.

[0215] Lactobacillus plantarum DSM 32131 (GOS42™) was deposited on 2 September 2015 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7 B, D-38124 Braunschweig, Germany by Probi AB.

[0216] Lactobacillus plantarum DSM 17852 (LB3e™) and Lactobacillus plantarum DSM 17853 (LB7c™) were deposited on 6 January 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, by Probac AB. All rights and duties in connection with microorganism deposits DSM 17852 and DSM 17853 were subsequently given to and accepted by Probi AB, who is now the depositor of the DSM 17852 and DSM 17853 strains.

[0217] Another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the administration of the at least one L. paracasei strain: a) improves endothelial function in the subject; and / or b) alleviates endothelial dysfunction in the subject; and / or c) improves lipid metabolism in the subject; and / or d) delays and / or reduces and / or prevents weight gain in the subject; and / or e) lowers triglycerides (optionally lowers triglyceride-rich lipoproteins) in the subject; and / or f) alleviates metabolic severity in the subject; and / or g) reduces cholesterol in the subject, preferably wherein the cholesterol is remnant cholesterol.

[0218] Terms such as "improves", "alleviates", "delays", "reduces", "prevents" and "lowers" may be relative to the specific parameter (e.g. endothelial function) had the subject not taken the at least one strain of L. paracasei. Alternatively, the terms may be relative to the specific parameter (e.g. endothelial) before the supplementation period and / or prior to receiving the at least one strain of L. paracasei, uses or methods of the of the invention.

[0219] By way of example, "improves" endothelial function means an improvement in endothelial function relative to the endothelial function had the subject not taken the at least one strain of L. paracasei. Alternatively, the improvement may be relative to endothelial function in the subject before the supplementation period and / or prior to receiving the at least one strain of L. paracasei in the aspects of the invention. Yet, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the administration of the at least one L. paracasei strain: a) supports endothelial function in the subject; and / or b) maintains endothelial function in the subject; and / or c) aids and / or assists repair of the gastrointestinal gut lining and / or gut wall lining in the subject.

[0220] Further, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the cardiometabolic disease is selected from a heart attack, a stroke, diabetes, insulin resistance, cardiovascular heart disease and non-alcoholic fatty liver disease.

[0221] By "heart attack", also known as a myocardial infarction (MI), we include when the flow of blood that brings oxygen to a part of the heart muscle suddenly becomes blocked. As a result, the heart can't get enough oxygen, leading to damage. If blood flow isn't restored promptly, the heart muscle begins to die. Symptoms of a heart attack include: chest pain (pressure, heaviness, tightness, or squeezing); pain in other areas (arms, jaw, neck, back, and abdomen); lightheadedness or dizziness; sweating; shortness of breath; nausea or vomiting; anxiety (similar to a panic attack); coughing and / or wheezing.

[0222] A stroke is a sudden impairment or loss of consciousness, sensation, and voluntary motion caused by a rupture or obstruction (such as a clot) in a blood vessel supplying the brain, resulting in permanent damage to brain tissue.

[0223] Still, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the cardiovascular disease is selected from a coronary heart disease, stroke, peripheral arterial disease and aortic disease.

[0224] Coronary heart disease (CHD), also known as ischemic heart disease or coronary artery disease, occurs when the blood supply to the heart muscle is blocked or interrupted due to a buildup of fatty substances (atherosclerotic plaque) in the coronary arteries. This condition can lead to symptoms like chest pain (angina), shortness of breath, and other related discomforts.

[0225] Peripheral arterial disease (PAD), also known as peripheral vascular disease (PVD), is a common condition where fatty deposits accumulate in the arteries, restricting blood supply to leg muscles. People with PAD may experience symptoms such as intermittent claudication (painful leg ache during exercise), hair loss on legs and feet, numbness, ulcers, and skin colour changes.

[0226] Another embodiment of the present invention relates to the at least one strain, the method or the use as described herein, wherein the risk factor associated with a cardiovascular disease or the risk factor associated with a cardiometabolic disease is selected from: a) reduced endothelial function; and / or b) increased body weight gain; and / or c) increased body mass index (BMI); and / or d) increased waist circumference; and / or e) increased blood pressure; and / or f) increased triglyceride levels; and / or g) increased total cholesterol; and / or h) increased remnant cholesterol; and / or i) increased low-density lipoprotein cholesterol (LDL); and / or j) decreased high-density lipoprotein cholesterol (HDL-c); and / or k) increased metabolic severity (e.g. increased metabolic severity score (MetS-Z); and / or l) reduced and / or impaired glucose metabolism; and / or m) increased / impaired fasting blood glucose; and / or n) insulin resistance or reduced insulin sensitivity.

[0227] A further embodiment of the present invention relates to the at least one strain, the method or the use as described herein, wherein the administration of the at least one L. paracasei strain: a) maintains normal blood cholesterol levels in a subject; and / or b) maintains healthy blood lipids / blood fats in a subject; and / or c) supports / promotes / maintains blood vessel function in a subject; and / or d) supports / promotes / maintains normal cholesterol levels.

[0228] The subject may be instructed to consume a therapeutically effective amount of the at least one probiotic strain according the method, the at least one strain or the use as described herein in combination with water, another aqueous solvent or a food product.

[0229] Yet, a further embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain is administered at a daily dose of from about 1 x 106to about 1 x 1014colony forming units (CFU) and / or about 1 x 109to about 1 x 1011CFU. In a preferred embodiment of the present invention, the daily dose of the at least one strain is 1 x 1010CFU.

[0230] It will be appreciated that a preferable daily dose may also be achieved by administration of more than one sub-dose, for example, by a twice daily administration of a unit dose comprising half of the preferable daily dose. Hence, the preferred ranges for the effective dose may also represent the preferred daily dosage to be achieved in whatever number of unit doses is practical.

[0231] If the at least one strain consists of more than one strain, such amounts represent the total CFU / dose of the combination of strains.

[0232] Still, a further embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain is administered daily and / or in a single unit dosage form.

[0233] The composition according to the invention may be a dietary supplement. By “dietary supplement” we include the meaning of a manufactured product intended to supplement the diet when taken by mouth, e.g. as a pill, capsule, tablet, or liquid. Dietary supplements may contain substances that are essential to life and / or those that have not been confirmed as being essential to life but may have a beneficial biological effect. When the composition according to the invention is in the form of a dietary supplement the carrier(s) to be added include those well known to a skilled person in the art, for example those given in Remington: The Science and Practice of Pharmacy, 19thed., vol. 1 & 2 (ed. Gennaro, 1995, Mack Publishing Company). Any other ingredients that are normally used in dietary supplements are known to a skilled person and may also be added conventionally together with the strain.

[0234] Another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain is administered for at least four weeks, at least eight weeks or at least twelve weeks.

[0235] Further, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain of Lacticaseibacillus paracasei and / or the at least one strain of Lactiplantibacillus plantarum is alive, dead or attenuated (e.g. a postbiotics and / or paraprobiotic and / or heat- inactivated), preferably wherein the at least one strain is alive. Still, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain is administered in the form of a composition comprising at least one pharmaceutically and / or nutritionally acceptable carrier, excipient and / or diluent material.

[0236] The at least one Lactobacillus plantarum strain according to any aspect of the invention may be present in a composition comprising at least one pharmaceutically and / or nutritionally acceptable carrier, diluent or excipient material. The composition may be as a solid or liquid formulation, and hence the at least one carrier may be a solid or a liquid, or may comprise both at least one solid component and at least one liquid component.

[0237] Examples of a suitable liquid carrier include water, milk, coconut water, fruit drinks and juices, milk substitutes (soya drink, oat drink, nut and other plant-based drinks), sparkling beverages, oil formulations including one or more of a nut or vegetable oil, such as coconut, rapeseed, olive, palm, corn / maize; glycerin, propylene glycol; and aqueous solvents.

[0238] Examples of a suitable solid carrier or excipient include maltodextrin, inulin, a cellulose such as microcrystalline cellulose (MCC), hydroxypropylmethylcellulose (HPMC) or hydroxypropylcellulose (HPC), sugar alcohols, high molecular weight polyethylene glycols, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato, tapioca or other vegetable starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0239] The carrier may be selected from a pharmaceutically acceptable carrier, excipient, or diluent; and a nutritionally acceptable i.e. food-grade carrier, excipient, or diluent material. For example, the carrier material may be a food.

[0240] Examples of suitable “pharmaceutically acceptable” carriers, excipients and diluents include those well known to a skilled person in the art, for example those given in Remington: The Science and Practice of Pharmacy, 19thed., vol. 1 & 2 (ed. Gennaro, 1995, Mack Publishing Company).

[0241] By “nutritionally acceptable” or “food-grade” we include carriers, ingredients and excipients that meet the ‘generally recognized as safe’ (GRAS) criteria. By “food” we include any substance for consumption to provide nutritional benefit or support for an organism. Examples of suitable food carriers include beverages (e.g. juices), dairy products (e.g. yoghurts, cheese, ice creams, infant formula and spreads such as margarine), dairy-alternative products (e.g. soy, nut or other plant-based drinks, yoghurts and spreads), cereal-based products (e.g. breads, biscuits, breakfast cereals, pasta and dry food bars such as health bars), and baby food (e.g. pureed fruit and / or vegetable).

[0242] The composition according to the invention may be a dry, non-fermented composition, a fermented composition, or a dry, fermented composition. Fermentation in this context particularly includes lactic acid fermentation by lactic acid bacteria in anaerobic conditions. In the case of a dry, non-fermented composition, substantially no fermentation takes place before ingestion by a subject, and so fermentation only takes place in the gastrointestinal tract after ingestion of the composition by a subject.

[0243] Hence, in some embodiments according to the invention, the composition is in the form of a food wherein the food is a cereal-based product, a dairy product, a juice drink, or a fermented food.

[0244] Examples of fermented foods include fermented milk products (such as yoghurt, kefir or lassi), fermented dairy-free milk alternatives (such as coconut milk kefir), fermented cereal-based products (such as oats, oatmeal, maize, sorghum, wheat), fermented vegetables (such as sauerkraut, kimchi, or pickles), fermented legumes or soybeans (such as natto or tempeh) and fermented tea (such as kombucha).

[0245] In use, the at least one strain or the composition comprising the at least one strain according to the invention may be mixed with a liquid or solid carrier before administration. For example, one may mix the strain or the composition thereof with a carrier comprising one or more liquids chosen from water, milk, coconut water, fruit drinks and juices, milk substitutes (soya drink, oat drink, nut and other plant-based drinks), sparkling beverages or some other aqueous solvent or drink prior to intake. Similarly, the at least one strain or the composition thereof may be mixed with a carrier consisting of one or more foods. Suitable food carriers include oatmeal carrier, barley carrier, fermented or non-fermented dairy products such as yoghurts, ice creams, milkshakes, fruit juices, beverages, soups, breads, biscuits, pasta, breakfast cereals, dry food bars including health bars, plant-based foods such as soy products, spreads, baby food, infant nutrition, infant formula, breast milk replacements from birth. Yet, another embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the carrier material is a food.

[0246] A further embodiment of the present invention relates to the method, the at least one strain or the use as described herein, wherein the at least one strain of Lacticaseibacillus paracasei is provided in a form selected from a solution, suspension, emulsion, tablet, granule, powder, capsule, lozenge, chewing gum, suppository, fast melt, or powder shot, preferably wherein the composition is provided in a capsule, fast melt or powder shot. Preferably, wherein the composition is provided in the form of capsule. Further, in a preferred embodiment of the present invention the composition is provided in the form of ca psule.

[0247] The at least one strain may be administered in the form of a powdered composition such as a fast-melt microbial composition, for example those described in WO 2017 / 060477, or in Probi's UK Patent Application 1708932.7 or Probi's publication WO 2018 / 224509 relating to Probi® Fast Melt technology. Where the powder is not in a fast-melt microbial composition, it may be suitable for being added to a food (e.g. yoghurt) or drink (e.g. water or milk) before ingestion.

[0248] Where the at least one strain is in the form of a powder, it would typically be filled in a sealed container, which provides an oxygen and moisture barrier in order to protect and maintain the viability of the bacteria in the composition. Hence, where the at least one strain is in the form of a powder, preferably the composition is packaged in sealed aluminium foil sticks, where each stick comprises one dose of the composition, i.e. one dose of the bacteria. Non-limiting examples of suitable containers include a stick, bag, pouch or capsule. In a preferred embodiment, the container is an aluminium foil or a polyethylene stick, which is typically sealed by welding. The stick is typically configured for easy tear opening. The stick may have a tear notch.

[0249] The at least one strain according to the invention may be formulated as a controlled- release solid dosage form, for example any of those described in WO 03 / 026687 and US Patent Nos. 8,007,777 and 8,540,980. The composition may be formulated as a layered dosage form, for example Probi's BlO-tract® technology including any of the layered dosage forms described in WO 2016 / 003870.

[0250] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the at least one strain (e.g. freeze-dried) in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.

[0251] Another embodiment of the present invention relates to a composition comprising at least one strain of Lacticaseibacillus paracasei for use as defined by the present invention as described herein.

[0252] Further, another embodiment of the present invention relates to a pharmaceutical composition comprising at least one strain of Lacticaseibacillus paracasei for use as defined by the present invention as described herein.

[0253] Yet, another embodiment of the present invention relates to a method comprising administering at least one strain of Lacticaseibacillus paracasei substantially as described herein.

[0254] Still, another embodiment of the present invention relates to at least one strain of Lacticaseibacillus paracasei for use substantially as described herein.

[0255] A further embodiment of the present invention relates to use of at least one strain of Lacticaseibacillus paracasei substantially as described herein.

[0256] Yet, a further embodiment of the present invention relates to use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use as substantially as described herein.

[0257] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "comprises", "preventing", "reducing", "supporting", "maintaining", "promoting", "aiding", "assisting", "provide", "providing", "ameliorating", "treating" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorth and method for referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All processes and methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as" and i.e.) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0258] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0259] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention.

[0260] EXAMPLES

[0261] Introduction

[0262] The purpose of the study was to investigate the effect of L. paracasei on alleviation of endothelial dysfunction in subjects with metabolic syndrome (MetS). The study was a randomized, double-blind and placebo-controlled trial among 130 participants with MetS, subjects were randomly assigned to placebo or L. paracasei 8700:2 for 12 weeks.

[0263] Summary

[0264] Background & aims:

[0265] Modulating microbial metabolism via probiotic supplementation has been proposed as an attractive strategy for the prevention of cardiometabolic diseases. Recently, Lacticaseibacillus paracasei (L. paracasei) was reported to alleviate metabolic disorders in murine models, however, its beneficial effects in humans remain to be determined. This study evaluated whether L. paracasei supplementation could improve endothelial function and cardiometabolic health in subjects with metabolic syndrome (MetS).

[0266] Methods:

[0267] In this randomized, double-blind and placebo-controlled trial among 130 participants with MetS, subjects were randomly assigned to placebo or L. paracasei 8700:2 (10 billion CFU) daily for 12 weeks. Endothelial function was measured by flow-mediated slowing, and cardiometabolic health was determined by both components and severity of MetS. Ideal compliance was defined as consumption no less than 70% of the capsules.

[0268] Results: 130 individuals (mean [SD] age, 45.97 [7.11] years; 95 men [73.1%]) were enrolled and randomized to L. paracasei (n = 66) or placebo control (n = 64). Compared to placebo, L. paracase / supplementation led to a greater reduction in remnant cholesterol (-0.16 mmol / L, 95%CI: -0.29 mmol / L to -0.02 mmol / L; P = 0.024). Such a reduction in remnant cholesterol was significantly associated with improvement in endothelial function (r = -0.23, P = 0.027). In subjects with an ideal compliance with trial protocol, L. paracasei treatment additionally lowered triglycerides, alleviated MetS severity and delayed weight gain. On the contrary, no obvious effect on insulin sensitivity or pancreatic beta-cell function was observed after L. paracasei intervention. Moreover, regarding safety and tolerability, no significant between-group difference in protocol-specified adverse events of interest was observed.

[0269] Conclusions:

[0270] L. paracasei supplementation enhanced endothelial function potentially through downregulating remnant cholesterol levels. Our study provides a feasible and safe strategy for the prevention of cardiometabolic diseases in subjects with severe dyslipidaemia and endothelial dysfunction.

[0271] Methods and materials

[0272] Study design

[0273] This was a 12-week randomized double-blinded placebo- controlled trial testing the effect of L. paracasei supplementation on cardiometabolic health in subjects with metabolic syndrome (MetS).. All participants provided written informed consents before randomization.

[0274] Recruitment and study participants

[0275] Adults aged between 30 and 65 years with MetS were recruited from the community health service center (Qishi Town, Guangdong, China) mainly through flyers and advertisement. Local residents who met the diagnosis of MetS according to electronic medical records were also invited by phone calls. Subjects with infections, any diet or medication that might interfere with metabolic homoeostasis and gut microbiota, especially antibiotics and probiotics within 4 weeks before recruitment, were excluded.

[0276] Inclusion criteria

[0277] Local residents aged between 30 to 65 years old with stable weight (<5% weight change over last 3 months) and presence of metabolic syndrome which was defined as having 3 or more metabolic abnormalities:

[0278] (1) Central obesity: waist circumference >90 cm in males or >80 cm in females;

[0279] (2) Elevated triglycerides: serum triglycerides >150 mg / dL (1.7 mmol / L);

[0280] (3) Reduced HDL cholesterol (HDL-C): serum HDL-C <40 mg / dL (1.03 mmol / L) in males or <50 mg / dL (1.29 mmol / L) in females;

[0281] (4) Elevated blood pressure: systolic blood pressure >130 mmHg or diastolic blood pressure >85 mmHg;

[0282] (5) Elevated fasting plasma glucose: fasting plasma glucose >100 mg / dL (5.6 mmol / L).

[0283] Exclusion criteria

[0284] (1) Presence of any systemic, cardiovascular diseases as well as infections within the previous month;

[0285] (2) Presence of any diet or medication which might interfere with metabolic homoeostasis and gut microbiota, especially antibiotics and probiotics 4 weeks before recruitment;

[0286] (3) Acute illness or current evidence of acute or chronic inflammatory diseases;

[0287] (4) Participation in any diet or weight loss program more than 2 times per week in the latest 3 months prior to recruitment;

[0288] (5) Mental illness rendering them unable to understand the nature, scope and possible consequences of the study;

[0289] (6) Women who were pregnant or intended to be pregnant during the study.

[0290] Randomization

[0291] Eligible participants were randomized into 2 groups (L. paracasei or placebo) via a computer-generated randomization list in a 1: 1 ratio, with a permuted block design (block sizes of 2 and 4). Capsules were pre-packaged in the same way according to randomization sequence which was prepared by an independent researcher who was not involved in this study. Thus, participants, field researchers and personnel responsible for data analysis remained blinded to treatment allocation until the database was unlocked and data analysis was completed.

[0292] Interventions

[0293] Participants were randomized to receive either L. paracasei capsule (£. paracasei 8700: 2, DSM 13434, Probi AB, Sweden) or placebo with the same ingredients except for L. paracasei. Several previous studies have evaluated the efficacy and safety of L. paracasei 8700: 2 on immunity-related outcomes, with dosages ranging from 1.0 x 109to 1.0 x 1010colony-forming units (CFU) per day. Generally, daily dosage up to 1.0 x 1010CFU of Lacticaseibacillus has been proved to be well tolerated in general population. Therefore, in this trial, 1.0 x 1010CFU per day was used to test the effect of L. paracasei 8700:2 on cardiometabolic health. The capsules were indistinguishable in terms of taste, colour and size. Eligible participants were instructed to take one capsule each day for 12 weeks, preferably at the same time, and return to the community healthcare centre every 4 weeks. To assess adherence, capsule boxes were collected every 4 weeks during clinic visits and counts of remaining capsules were completed by an independent person. Low compliance was defined as consumption of less than 70% of the capsules. During the study period, any adverse effects and unfavourable responses related to the trial were every 2 weeks by trained staffs. Additionally, participants in both groups were advised to maintain their lifestyles including habitual diet and physical activity during the study period.

[0294] Data collection

[0295] Collection of data during each follow-up visit was shown in Fig. 1A. Briefly, face-to- face interview with a validated questionnaire, physical examination and biological measurements were performed at baseline and every 4 weeks during the study period. Blood samples were collected between 8:00 to 9:00 after an overnight fast for 10-12 hours at baseline and each follow-up visit, to minimize potential influences from circadian rhythms. 75-g oral glucose tolerance test was conducted at baseline and after 12-week intervention and blood samples were taken for the assessment of plasma glucose and insulin levels at 0, 60 and 120 min. Samples were processed immediately and all biochemical measurements were performed within 3 hours in the central laboratory of community healthcare centre after collection via an automated analyzer (Mindray BS600, Mindray, Shenzhen, China), except for insulin and glycated haemoglobin (HbAic). Insulin was measured by an enzyme-linked immunosorbent assay (10-1113- 01, Mercodia, Uppsala, Sweden), and HbAic was determined with high performance liquid chromatography by technicians blinded to the study design. Body weight was measured by a body composition monitor (BC-601, TANITA, Tokyo, Japan) and blood pressure was measured with an automated electronic sphygmomanometer (HEM- 7136, OMRON, Dalian, China) after resting for more than 5 min. All these measurements were performed twice with at least 2-min interval and reported as an average value. Dietary intake was collected by 3-day food record (2 separate weekdays and 1 weekend), and analysed by NutriStar software (Zhending Inc, Shanghai, China). Physical activity was monitored by international physical activity questionnaire short form and evaluated as metabolic equivalents and sedentary time, at baseline and every 4 weeks during the study period.

[0296] Outcome measures

[0297] The primary outcome was alteration in flow-mediated slowing (FMS), a practical, reliable and sensitive alternative technique for the assessment of endothelial function in community residents, measured by Vicorder (Skidmore medical, Bristol, UK) at baseline and week 12. A lower value of FMS indicates a worse endothelial function.

[0298] Pre-specified secondary outcomes included: (1) changes in lipid profiles, including triglyceride (TG), total cholesterol (TC), low- density lipoprotein cholesterol (LDL-c), high- density lipoprotein cholesterol (HDL-c) and remnant cholesterol; (2) decrease in the number of MetS components and MetS severity reflected by metabolic syndrome severity z score (MetS-Z); (3) reduction in body weight, body mass index (BMI), and waist; and (4) improve- ments in blood pressure, glucose metabolism and insulin sensitivity, including fasting or postprandial glucose, HbAic, Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), Homeostasis Model Assessment of b-cell function (HOMA-b), Matsuda index and Stumvoll insulin secretion. Remnant cholesterol was calculated as TC minus HDL-c minus LDL- c, as previously described.

[0299] Sample size and statistical analyses

[0300] On the basis of an estimated 41% improvement in endothelial function from baseline to 12 weeks, a sample size of 36 per group would provide 80% power to detect the effect of L. paracasei at a two-sided significance of 5%. Assuming a 10% loss of follow up, 40 subjects per group was needed. However, in case of a high drop- out rate due to the pandemic of COVID-19, a total of 130 participants were enrolled.

[0301] Intention-to-treat (ITT) analysis was applied for primary outcomes and pre-specified secondary outcomes, which included all participants who were randomized regardless of any deviation from trial protocol. Considering that compliance might affect the effect of probiotics on metabolic health, per-protocol analysis was further conducted which included only participants with adequate adherence to the assigned regimen. Data from the time when the participants experienced a major protocol deviation was excluded in perprotocol analysis. Additionally, safety analysis was performed in all participants who received at least one capsule. Change in FMS from baseline to week 12 was analysed with a generalized estimating equation model, in order to account for missing values and correlation among repeated measurements. The model included treatment as fixed effects, visit and treatment-by-visit interaction, along with covariates of age, gender and the baseline value of each participant. Least square means of change and two-sided 95%CIs for each group and the pairwise comparisons between two groups were determined. The same approach was employed for all secondary outcomes, including changes in lipid profiles, MetS severity, body shape, blood pressure, glucose metabolism, insulin sensitivity and beta-cell function from baseline to week 4, 8 and 12. Subgroup analyses was performed in subjects with severe endothelial dysfunction, which was defined as a baseline FMS reading less than 17, the median value of study population in Southern China (data not shown). Characteristics of the study participants were expressed as mean (SD) for normally distributed continuous variables, median [25th, 75th percentile] for skewed variables, and n (%) for category variables. Adjustments for multiple comparisons were made using least significant difference for all outcomes and no imputation of missing data was performed. All data were analysed using SPSS 25.0 (IBM SPSS Statistics) and R 4.0.5 with a significance level at P < 0.05 (two-sided).

[0302] Results

[0303] Example 1 - Study population and monitoring of lifestyles during the study period

[0304] Out of the 254 subjects undergoing screening, a total of 130 subjects with MetS (mean [SD] age: 45.97 [7.11]; 95 men [73.1%]) were enrolled and randomly assigned to L. paracasei or placebo control. Most characteristics including endothelial function, and the components and severity of MetS were similar between the two groups, except for a higher proportion of hypertriglyceridemia and higher levels of remnant cholesterol in subjects allocated to L. paracasei intervention (Table 1). A total of 103 participants completed the 12- week trial and attended the final clinic visit, with 55 (83.3%) in L. paracasei and 48 (75.0%) in placebo group, respectively. Withdrawal or loss of follow-up due to busy schedule was the most common reason for study discontinuation in both groups. The median adherence to allocated treatments didn't differ (87.3% for L. paracasei vs. 87.5% for placebo), and thus resulting in 48 and 42 subjects in L. paracasei and placebo group respectively for per- protocol analysis (Fig. IB). Similarly, baseline characteristics showed no significant difference between the two groups enrolled in per-protocol analysis (Table 2). Considering that changes in lifestyles may have a crucial impact on cardiovascular health, physical activity and diet intake were monitored throughout the study. Except for a slightly higher intake of fat in subjects allocated to L. paracasei at baseline, there was no significant difference in physical activity and dietary intake between the two groups throughout the study period (Table 3).

[0305] Table 1 - Characteristics of study participants at enrolment.

[0306]

[0307] Data were presented as n (%), mean (SD) or median (interquartile range), as appropriate. BMI = body mass index. DBP = diastolic blood pressure. FMS = flow-mediated slowing. HbAlc = glycated hemoglobin. HDL-c = high-density lipoprotein cholesterol. HOMA-IR = homeostasis model assessment insulin resistance. HOMA-p = homeostasis model assessment of 3-cell function. LDL-c = low-density lipoprotein cholesterol. L. paracasei = Lacticaseibacillus paracasei 8700:2. SBP = systolic blood pressure. TC = total cholesterol. TG = triglyceride.

[0308] Table 2 - Baseline characteristics of the study participants for per-protocol analysis

[0309]

[0310] Data were presented as n (%), mean (SD) or median (interquartile range) as appropriate. BMI=body mass index. DBP=diastolic blood pressure. FMS=flow-mediated slowing. HbAlc=glycated hemoglobin. HDL-c=high-density lipoprotein cholesterol. HOMA-IR=homeostasis model assessment insulin resistance. HOMA-[3=homeostasis model assessment of [3-cell function. LDL-c=low-density lipoprotein cholesterol. L. paracasei= Lacticaseibacillus paracasei 8700:2. SBP=systolic blood pressure. TC=total cholesterol. TG=triglyceride.

[0311] 'able 3 - Physical activity and dietary intake during the study period

[0312] aMean (SD) was obtained from generalised estimating equation.bPairwise comparison with generalized estimating equation.

[0313] L. paracasei=Lacticaseibacillus paracasei 8700:2. MET = metabolic equivalent.

[0314] Example 2 - Effect of L. paracasei on alleviation of endothelial dysfunction in subjects with metabolic syndrome

[0315] For the primary outcome, 12-week intervention of L. paracasei significantly improved FMS with an adjusted mean change from baseline of 3.13% (95%CI: 0.73%e5.53%; P1 / 4 0.011) while the placebo group showed a non-significant increase, resulting in a between- group difference of 1.28% (95%CI: -2.10%e4.65%; P1 / 4 0.459; Fig. 2). In per-protocol analysis, mean change from baseline was 3.55% (95%CI: 0.94%e6.15%; P1 / 4 0.008) for L. paracasei and 2.30% (95%CI : -0.31%e4.90%; P1 / 4 0.084) for placebo, resulting in a difference of 1.25% (95%CI : -2.43%e4.94%; P V40.506; Fig. 2).

[0316] Considering that the participants are generally healthy, subgroup analysis was further performed across FMS level. When restricted to subjects with worse baseline endothelial function, the difference between the L. paracasei and placebo group was significant both in ITT (4.61%; 95%CI: 0.51%e8.72%; P V40.028) and per-protocol population (5.04%; 95%CI: 0.32%e9.75%; P V40.036; Fig. 2).

[0317] Although probiotics are viable non-pathogenic microorganisms able to confer health promoting effects, as a live bacterium, it may also have the possibility of eliciting unfavourable or unexpected immune response, especially in subjects with impaired metabolism. Therefore, the safety and tolerance of L. paracasei supplementation was monitored throughout the study period.

[0318] During the 12-week intervention, no serious adverse events, such as allergy or bacteraemia, were reported and no significant differences in liver enzymes, or the counts or composition of white blood cells were found between L. paracasei and placebo (Table 4), suggesting that daily supplementation of L. paracasei was safe in subjects with MetS. The adverse events reported by 7 participants were evenly distributed between two groups (7.58% in L. paracasei and 3.13% in placebo group, P = 0.441). The majority of the adverse events was related to gastrointestinal discomfort which can be tolerated and released shortly, such as nausea, diarrhoea, flatulence and stomach ache. . No serious adverse events, such as allergy or bacteremia, were reported and no between-group difference in liver enzymes, counts or composition of white blood cells was found during the 12-week trial, suggesting that daily supplementation of L. paracasei was safe in subjects with MetS.

[0319] Example 3 - Effect of L. paracasei on the improvement of lipid metabolism and its association with endothelial function

[0320] Introduction The purpose of the study was to investigate the effect of L. paracasei on the improvement of lipid metabolism and its association with endothelial function. Subjects with MetS, were randomly assigned to placebo or L. paracasei 8700:2 for 12 weeks.

[0321] Result

[0322] This study found that L. paracasei intervention substantially improved lipid profiles, especially for the reduction of triglyceride-rich lipoproteins. In ITT population, L. paracasei treatment showed a modest but constant decrease in TG with a mean change from baseline to 12 week of -0.43 mmol / L (95%CI : -0.88 mmol / L to 0.02 mmol / L) while the level of TG tended to rise for placebo group (Fig. 3A). As for remnant cholesterol, a cholesterol content of triglyceride-rich lipoproteins which represents an essential residual cardiovascular risk, it demonstrated a constant decrease in subjects allocated to L. paracasei while remained almost unchanged in participants randomized to the placebo group. Despite a markedly higher level of remnant cholesterol at baseline in subjects allocated to L. paracasei, compared to placebo control, L. paracasei intervention still led to a greater reduction in remnant cholesterol (net difference: -0.16 mmol / L, 95%CI: -0.29 mmol / L to -0.02 mmol / L; P1 / 4 0.024; Fig. 3B), after correcting for age, sex and baseline levels.

[0323] The amelioration of triglyceride-rich lipoproteins was more obvious in individuals with a high compliance with the trial protocol, as reflected by a significant between-group difference of -0.78 mmol / L for TG (95%CI: -1.51 mmol / L to -0.05 mmol / L; P1 / 4 0.037; Fig. 3C), and -0.22 mmol / L for remnant cholesterol (-0.36 mmol / L to -0.07 mmol / L; P1 / 4 0.003; Fig. 3D) in per-protocol analysis. Considering the close association of remnant cholesterol with cardiovascular health, the correlation between changes in FMS and remnant cholesterol from baseline to week 12, was next explored and an inverse association between increased remnant cholesterol and improved endothelial function was found, after adjustment for age, gender and baseline values (ITT population: r = -0.23, P = 0.027, Fig. 3E; per-protocol population: r = -0.24, P = 0.026, Fig. 3F). As for other lipid parameters, such as TC, HDL-c and LDL-c, no significant difference between L. paracasei and placebo was found after 12-week intervention (Fig. 4). Table 4 - Change in liver function and white blood cells from baseline to week 12

[0324]

[0325] Mean difference between-group and P values are from generalized estimating equation model with treatment, visit and treatment-by-visit as factors, gender, age and the baseline value as covariates. Two-sided 95% Cis and P values for each treatment group by pairwise comparisons were determined. ALT=alanine aminotransferase; AST=aspartate aminotransferase.

[0326] Example 4 - Effect of L. paracasei on amelioration of metabolic disorders.

[0327] Introduction

[0328] The purpose of the study was to investigate the effect of L. paracasei on amelioration of MetS. Subjects with MetS, were randomly assigned to placebo or L. paracasei 8700:2 for 12 weeks.

[0329] Results

[0330] In ITT population, significant improvement in MetS was only found in individuals allocated to L. paracasei treatment, both in terms of MetS component numbers and severity (Fig. 5A and B). When further restricted to participants with good compliance, there were significant between-group differences in the amelioration of MetS components (-0.48, 95%CI: -0.94 to -0.02, P = 0.041, Fig. 5C) and severity (MetS-Z: -0.15, 95%CI: -0.29 to -0.01, P = 0.034, Fig. 5D). Notably, the ameliorative effects of L. paracasei on MetS might be driven by its delay in body weight gain (Fig. 5E-H), which was much more prominent in individuals with an ideal adherence to the study protocol, as reflected by a between- group difference of -0.67 kg (95%CI: -1.35 kg to 0.00 kg, P = 0.051, Fig. 5G) and -0.29 (95%CI: -0.54 to -0.03, P = 0.029, Fig. 5H) for body weight and BMI, respectively. On the contrary, as for other components of MetS, such as central obesity, raised blood pressure, disturbance in glucose metabolism and insulin resistance, no significant between-group differences were found either in ITT or PP analysis (Table 5).

[0331] Table 5 - Change in obesity, blood pressure, glucose metabolism and insulin sensitivity from baseline to week 12

[0332]

[0333] Mean difference between-group and P values are from generalized estimating equation model with treatment, visit and treatment-by-visit as factors, gender, age and the baseline value as covariates. Two-sided 95% Cis and P values for each treatment group by pairwise comparisons were determined. DBP=diastolic blood pressure. FPG=fasting plasma glucose. HOMA-IR=homeostasis model assessment insulin resistance. HOMA- [3= homeostasis model assessment of 3-cell function. L. paracasei= Lacticaseibacillus paracasei 8700:2. SBP=systolic blood pressure.

[0334] Discussion and Conclusion

[0335] The Examples above provides evidence that daily intake of L. paracasei may be an attractive approach to lower remnant cholesterol and its associated cardiovascular risk. In addition to the lipid-lowering effect, supplementation of L. paracasei for 12 weeks demonstrated a notable ameliorative effect on MetS, as evidenced by both a reduced number of metabolic disorder and its severity (Fig. 5A-D). Such an alleviation in MetS might be mainly driven by a reduced body weight gain during the intervention since significant body gain was observed in subjects allocated to the placebo group (Fig. 5E-H).

[0336] This randomized, double-blind, placebo-controlled trial suggests that dietary L. paracasei 8700:2 supplementation has favourable effects on endothelial function, potentially though reducing remnant cholesterol. The study demonstrates that L. paracasei supplementation is an effective, feasible and safe strategy for the prevention of cardiometabolic diseases in subjects with severe endothelial dysfunction and dyslipidaemia.

[0337] Collectively, the above findings suggest that probiotics function as a useful tool for both primary and secondary prevention of cardiovascular diseases.

Claims

CLAIMS1. A method for reducing and / or preventing weight gain in a subject, comprising administering to a subject in need thereof, an effective dose of at least one strain of Lacticaseibacillus paracasei.

2. At least one strain of Lacticaseibacillus paracasei for use in reducing and / or preventing weight gain in a subject in need thereof.

3. Use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

4. Non-therapeutic use of at least one strain of Lacticaseibacillus paracasei for reducing and / or preventing weight gain in a subject.

5. Use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in reducing and / or preventing weight gain in a subject in need thereof.

6. A method for the prevention and / or amelioration and / or treatment of metabolic syndrome in a subject, comprising administering an effective dose of at least one strain of Lacticaseibacillus paracasei to a subject in need thereof.

7. At least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

8. Use of at least one strain of Lacticaseibacillus paracasei for the manufacture of a medicament for use in preventing and / or ameliorating and / or treating metabolic syndrome in a subject in need thereof.

9. A method for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

10. At least one strain of Lacticaseibacillus paracasei for improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

11. Use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use in improving cardiometabolic health and / or cardiovascular health in a subject in need thereof.

12. A method for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in needthereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

13. At least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

14. Use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiometabolic disease and / or for reducing a risk factor associated with a cardiometabolic disease in a subject in need thereof.

15. A method for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof, comprising administering to a subject in need thereof an effective dose of at least one strain of Lacticaseibacillus paracasei.

16. At least one strain of Lacticaseibacillus paracasei for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof.

17. Use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for preventing and / or ameliorating a cardiovascular disease and / or for reducing a risk factor associated with a cardiovascular disease in a subject in need thereof.

18. The method according to any one of claims 1, 6, 9, 12 or 15, the at least one strain according to any one of claims 2, 7, 10, 13 or 16, or the use according to any one of claims 3-5, 8, 11, 14 or 17, wherein the subject has metabolic syndrome.

19. The method according to any one of claims 1, 6, 9, 12, 15 or 18, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18, or the use according to any one of claims 3-5, 8, 11, 14, 17 or 18, wherein the subject is a mammal, preferably wherein the subject is human.

20. The method according to any one of claims 1, 6, 9, 12, 15, 18 or 19, the at least one strain according to any one of claims 2, 7, 10, 13, 16, 18 or 19, or the use according to any one of claims 3-5, 8, 11, 14 or 17-19, wherein the at least one strain of Lacticaseibacillus paracasei comprises Lacticaseibacillus paracasei DSM 13434 or Lacticaseibacillus paracasei DSM 13432.

21. The method according to any one of claims 1, 6, 9, 12, 15 or 18-20, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-20, or the use according toany one of claims 3-5, 8, 11, 14 or 17-20, wherein the at least one strain of Lacticaseibacillus paracasei is Lacticaseibacillus paracasei DSM 13434.

22. The method according to any one of claims 1, 6, 9, 12, 15 or 18-21, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-21, or the use according to any one of claims 3-5, 8, 11, 14 or 17-21, wherein the at least one strain is Lacticaseibacillus paracasei DSM 13434 in combination with at least one strain of Lactiplantibacillus plantarum.

23. The method according to any one of claims 1, 6, 9, 12, 15 or 18-22, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-22, or the use according to any one of claims 3-5, 8, 11, 14 or 17-22, wherein the at least one strain of Lactiplantibacillus plantarum is selected from Lactiplantibacillus plantarum DSM 6595, Lactiplantibacillus plantarum DSM 9843, Lactiplantibacillus plantarum DSM 15312, Lactiplantibacillus plantarum DSM 15313, Lactiplantibacillus plantarum DSM 15316, Lactiplantibacillus plantarum DSM 32131, Lactiplantibacillus plantarum DSM 17852 and Lactiplantibacillus plantarum DSM 17853.

24. The method according to any one of claims 1, 6, 9, 12, 15 or 18-23, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-23, or the use according to any one of claims 3-5, 8, 11, 14 or 17-23, wherein the at least one strain is Lacticaseibacillus paracasei DSM 13434 in combination with Lactiplantibacillus plantarum DSM 9843.

25. The method according to any one of claims 1, 6, 9, 12, 15 or 18-24, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-24, or the use according to any one of claims 3-5, 8, 11, 14 or 17-24, wherein the administration of the at least one L. paracasei strain: a) improves endothelial function in the subject; and / or b) alleviates endothelial dysfunction in the subject; and / or c) improves lipid metabolism in the subject; and / or d) delays and / or reduces and / or prevents weight gain in the subject; and / or e) lowers triglycerides (optionally lowers triglyceride-rich lipoproteins) in the subject; and / or f) alleviates metabolic severity in the subject; and / or g) reduces cholesterol in the subject, preferably wherein the cholesterol is remnant cholesterol.

26. The method according to any one of claims 1, 6, 9, 12, 15 or 18-25, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-25, or the use according to any one of claims 3-5, 8, 11, 14 or 17-25, wherein the administration of the at least one L. paracasei strain: a) supports endothelial function in the subject; and / or b) maintains endothelial function in the subject; and / or c) aids and / or assists repair of the gastrointestinal gut lining and / or gut wall lining in the subject.

27. The method according to any one of claims 9, 12 or 18-26, the at least one strain according to any one of claims 10, 13 or 18-26, or the use according to any one of claims 11, 14 or 18-26, wherein the cardiometabolic disease is selected from a heart attack, a stroke, diabetes, insulin resistance, cardiovascular heart disease and non-alcoholic fatty liver disease.

28. The method according to any one of claims 9, 15 or 18-27, the at least one strain according to any one of claims 10, 16 or 18-27, or the use according to any one of claims 1, 17 or 18-27, wherein the cardiovascular disease is selected from a coronary heart disease, stroke, peripheral arterial disease and aortic disease.

29. The at least one strain for use according to any one of claims 12 or 18-28, the method according to any one of claims 13 or 18-28 or the use according to any one of claims 14 or 18-28, wherein the risk factor associated with a cardiovascular disease or the risk factor associated with a cardiometabolic disease is selected from: a) reduced endothelial function; and / or b) increased body weight gain; and / or c) increased body mass index (BMI); and / or d) increased waist circumference; and / or e) increased blood pressure; and / or f) increased triglyceride levels; and / or g) increased total cholesterol; and / or h) increased remnant cholesterol; and / or i) increased lowdensity lipoprotein cholesterol (LDL); and / or j) decreased high-density lipoprotein cholesterol (HDL-c); and / or k) increased metabolic severity (e.g. increased metabolic severity score (MetS-Z); and / or l) reduced and / or impaired glucose metabolism; and / or m) increased / impaired fasting blood glucose; and / orn) insulin resistance or reduced insulin sensitivity.

30. The method according to any one of claims 1, 6, 9, 12, 15 or 18-29, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-29, or the use according to any one of claims 3-5, 8, 11, 14 or 17-29, wherein the administration of the at least one L. paracasei strain: a) maintains normal blood cholesterol levels in a subject; and / or b) maintains healthy blood lipids / blood fats in a subject; and / or c) supports / promotes / maintains blood vessel function in a subject; and / or d) supports / promotes / maintains normal cholesterol levels.

31. The method according to any one of claims 1, 6, 9, 12, 15 or 18-30, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-30, or the use according to any one of claims 3-5, 8, 11, 14 or 17-30, wherein the at least one strain is administered at a daily dose of from about 1 x 106to about 1 x 1014colony forming units (CFU) and / or about 1 x 109to about 1 x 10nCFU.

32. The method according to any one of claims 1, 6, 9, 12, 15 or 18-31, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-31, or the use according to any one of claims 3-5, 8, 11, 14 or 17-31, wherein the daily dose of the at least one strain is 1 x 1010CFU.

33. The method according to any one of claims 1, 6, 9, 12, 15 or 18-32, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-32, or the use according to any one of claims 3-5, 8, 11, 14 or 17-32, wherein the at least one strain is administered daily and / or in a single unit dosage form.

34. The method according to any one of claims 1, 6, 9, 12, 15 or 18-33, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-33, or the use according to any one of claims 3-5, 8, 11, 14 or 17-33, wherein the at least one strain is administered for at least four weeks, at least eight weeks or at least twelve weeks.

35. The method according to any one of claims 1, 6, 9, 12, 15 or 18-34, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-34, or the use according to any one of claims 3-5, 8, 11, 14 or 17-34, wherein the at least one strain of Lacticaseibacillus paracasei and / or the at least one strain of Lactiplantibacillus plantarum is alive, dead or attenuated (e.g. a postbiotics and / or paraprobiotic and / or heat- inactivated), preferably wherein the at least one stain is alive.

36. The method according to any one of claims 1, 6, 9, 12, 15 or 18-35, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-35, or the use according to any one of claims 3-5, 8, 11, 14 or 17-35, wherein the at least one strain is administered in the form of a composition comprising at least one pharmaceutically and / or nutritionally acceptable carrier, excipient and / or diluent material.

37. The method according to any one of claims 1, 6, 9, 12, 15 or 18-36, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-36, or the use according to any one of claims 3-5, 8, 11, 14 or 17-36, wherein the carrier material is a food.

38. The method according to any one of claims 1, 6, 9, 12, 15 or 18-37, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-37, or the use according to any one of claims 3-5, 8, 11, 14 or 17-37, wherein the at least one strain of Lacticaseibacillus paracasei is provided in a form selected from a solution, suspension, emulsion, tablet, granule, powder, capsule, lozenge, chewing gum, suppository, fast melt, or powder shot, preferably wherein the composition is provided in a capsule, fast melt or powder shot.

39. The method according to any one of claims 1, 6, 9, 12, 15 or 18-38, the at least one strain according to any one of claims 2, 7, 10, 13, 16 or 18-38, or the use according to any one of claims 3-5, 8, 11, 14 or 17-38, wherein the composition is provided in the form of capsule.

40. A composition comprising at least one strain of Lacticaseibacillus paracasei for use as defined in any one of claims 2, 7, 10, 13, 16 or 18-39.

41. A pharmaceutical composition comprising at least one strain of Lacticaseibacillus paracasei for use as defined in any one of claims 2, 7, 10, 13, 16 or 18-39.

42. A method comprising administering at least one strain of Lacticaseibacillus paracasei substantially as described herein.

43. At least one strain of Lacticaseibacillus paracasei for use substantially as described herein.

44. Use of at least one strain of Lacticaseibacillus paracasei substantially as described herein.

45. Use of at least one strain of Lacticaseibacillus paracasei in the manufacture of a medicament for use as substantially as described herein.

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