Effect of Sour Creams Fermented by Limosilactobacillus fermentum AG8 and Lactiplantibacillus plantarum AG9 on Mice

Introduction

Sour cream, also fermented cream, is a type of dairy product with a unique, slightly sour taste, which is produced from high-quality cream as a raw material and a complex of mesophilic lactic acid bacteria (LAB).¹ Sour cream is a traditional and popular product in Russia and the other Slavic countries, it is produced in various fat content from 10 to 30%. Sour cream with 15% fat content is the most popular.^1,2 In various countries, sour cream is used as a sauce for soups, vegetable salads, meat, fish, flour dishes such as ravioli, and as a base for cake cream or as an ingredient in baked products.²

Fermentation is an established technique that is utilized in the preservation of valuable dairy products, with the aim of prolonging shelf life. In addition to its function in prolonging the shelf life of products, fermentation can also contribute towards a distinctive flavour and texture.³ Cream is the fat fraction of all types of milk, can be obtained from different animals by separating from the water fraction, so the fat content of sour cream is higher than other dairy products. Sour cream is traditionally fermented using starter cultures composed mainly of lactic acid bacteria (LAB), such as Lactococcus lactis subsp. lactis and L. lactis subsp. Cremoris.^4-6 These strains are characterized by their ability to synthesize elevated levels of aromatic compounds, which play a key role in developing the typical flavor and sensory properties of the final product.⁷ The process of forming low molecular weight substances such as aldehydes, alcohols, ketones is associated with the synthesis of enzymes for hydrolysing proteins, lipids and other organic substances during the fermentation of dairy raw materials. Besides lactococci, sour cream can be fermented by Leuconostoc spp. which, due to their heterofermentative nature, can release carbon dioxide, volatile compounds and organic acids that give fermented cream flavor and texture.^8,9 These aroma and flavors of sour cream makes it popular as an appetizer, salad dressing, a base for sauces.^10,11

However, the high fat content of sour cream limits its consumption in large quantities. The functional value of sour cream can be improved by using only probiotic bacteria¹² or by incorporating non-starter probiotic bacteria into a traditional sour cream starter.¹³ A popular strategy today is the use of non-starter probiotic bacteria as part of fermentation-derived products.¹⁴ The researchers are considering lactobacilli as a bacterial culture that can be used as a probiotic culture for the production of sour cream.¹³ Lactobacilli of various species are being tested for use in dairy products: Lactobacillus acidophilus,¹⁵ Lactiplantibacillus plantarum,^16,17 Lactobacillus rhamnosus,¹⁸ Lactobacillus helveticus.¹⁹

A considerable number of studies are available which address the use of probiotics in dairy products,^20,21 including in products high in milk fat.^22,23 However, the number of research papers focusing on sour cream is comparatively low. Researchers are primarily focused on studying the regularities of changes in rheology, texture, and the state of milk fat, as well as changes in the composition of aromatic compounds in sour cream.^1,12,24 There are limited studies in existence that address the effects of sour cream or fermented cream containing probiotic bacteria on mammals.

In the previous section, the objective was to elucidate the distinctive effects of sour cream made with traditional starter cultures (Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetilactis) and sour cream obtained by fermentation with probiotic lactic acid bacteria on mice. The selection of Limosilactobacillus fermentum AG8 and Lactiplantibacillus plantarum AG9 was made on the basis of their well-documented probiotic properties.²⁵ These strains have probiotic properties, are capable of fermenting milk and metabolic products have antioxidant properties.^26-28 In addition, L. fermentum strain AG8 was found to have high antibacterial activity.²⁹

Material and Methods

Strains and Fermented Cream?

The experiment utilized cream with a fat content of 15% (Valio®, Russia). To prepare the fermented cream, commercial starter cultures were employed, including Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetilactis (Lactosintez LLC, Moscow, Russia), strains Limosilactobacillus fermentum AG8 and Lactiplantibacillus plantarum AG9 were isolated from the silage and described earlier like probiotic strains.²⁵ These strains were cultivated on de Man, Rogosa, and Sharpe (MRS) medium or MRS-agar (HiMedia, India). The following sour cream samples were prepared for in vivo experiments: Fer_cream – classic sour cream fermented using commercial L. cremoris and L. diacetilactis starter cultures; Fer.cream_AG8 – fermented cream obtained by fermentation with L. fermentum AG8; Fer.cream_AG9 – fermented cream obtained by fermentation with L.plantarum AG9.

The sour cream samples were obtained as follows. For sour cream production, lactic acid bacteria (LAB) precultures were grown in sterile skim milk at 36°C for 16 h. The resulting precultures, with a volume of 5 ml, were added to the 100 ml of cream containing 15% fat. The mixture was then subjected to a fermentation process at a temperature of 32°C for a period of 14 hours. Following this, the sample underwent a stabilization step at a temperature of 4°C for a duration of 16 hours. Subsequent to this stabilization phase, each sample was stored at a temperature of 4°C and utilized for the purpose of animal feeding over a 21-day period. A fresh batch of fermented cream was prepared every 5 days to ensure freshness.

Animals

White laboratory mice (male) were used in this study. Twenty-eight mice, 6-7 weeks old (the mice weighed in at 12-14 g), were obtained from the Kazan Federal University, Kazan, Russia. The study was approved by the local ethical committee of Kazan Federal University (protocol No. 40 of March 9, 2023), where the research was conducted. All mice were kept at 20-24 º C in a room with 60-65% humidity and a light/dark cycle of 12 h, given free access to standard food and tap water for one week.

The mice were randomly divided into four groups of 7 individuals each, the following experimental studies were performed while varying the food supplementation:

(Control) group with normal diet received the basic feed and sterile water. Commercial mouse feed (DeltaFids, BioPro Company, Novosibirsk, Russia) was used (metabolizable energy – 2500 kcal/kg, crude protein – 19 %. Composition: two-component grain mixture, high-protein components (vegetable and animal proteins), vegetable oil, amino acids, organic acids, vitamin and mineral complex, fiber);

(Fer_cream) normal diet + fermented cream (1 g/animal per day);

(Fer.cream_AG8) normal diet + fermentum AG8 fermented cream (1 g/animal);

(Fer.cream_AG9) normal diet + plantarum AG9 fermented cream (1 g/animal).

Throughout the study period, body weights were recorded on a weekly basis at 7:00 am. After 21 days, blood samples from the animals were collected into disposable tubes with separation gel. The tubes were then put into a centrifuge (DM0506, Dlab, China) at 3500 rpm for 10 minutes. The spleen, heart, and both kidneys together, as well as the liver, were weighed for each animal. The organ index was determined as a percentage of the animal’s weight. Blood samples for serum lipid and blood morphology analysis were taken from the coronary artery after 21 days. Blood analysis was performed using an Abacus Junior 5 VET haematology analyser (Diatron Messtechnik GmbH, Austria). Blood samples were stored in a refrigerator at 2-4°C for no more than 1-2 hours prior to analysis. Biochemical parameters, including total cholesterol (TC), high-density lipoprotein (HDLP), low-density lipoprotein (LDLP), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein and bilirubin, were determined using a ChemWell2902 device (Awareness Technology, USA) and kits or reagents from Spinreact S.A. (Spain).

Microbiological Analyses

The contents of the large intestine (faeces) were analysed in this study. For this purpose, after autopsy, faeces were scraped into sterile plastic tubes, placed on ice and seeded onto appropriate media within 6 h. Numbers of different groups of microorganisms were analyzed by serial dilutions, the following nutrient media were used: for LAB (lactobacilli) – MRS agar (Himedia, India), total bacteria – nutrient agar SPA (Microgen, Russia), coli-forming bacteria and E. coli – Endo medium (Microgen, Russia), yeast – Saboura medium (Microgen, Russia). Cultivation after sowing was carried out at 36 º C for 1-5 days, depending on the species of microorganisms.

Statistical Analysis

All analyses were performed in three repetitions. Statistical significance of the results was determined at P < 0.05. Visualization of principal component analysis (PCA) and correlation analysis was performed on a two-dimensional P1/P2 map using OriginPro software v.8, which allowed us to identify relationships between variables at a significance level of P < 0.05.

Results

Organ Indices and Hematologic Indices

The addition of a moderate amount of sour cream to the diet of mice resulted in a slight decrease in body weight compared to the control. This effect may be due to an adjustment in lipid metabolism (Table 1). Compared to the control group, the organ indices (heart, kidneys, liver) were lower in the experimental groups than in the control group. Spleen index was lower in sour cream than in control, but consumption of L.fermentum AG8 and L.plantarum AG9 cream resulted in an increase in spleen index.

Table 1: Absolute weight gains of mice, the organ indexes over 21 days of feeding in an experiment with the introduction of sour cream into a balanced diet (mean values ± SD, n = 7).

With the exception of leukocytes and lymphocyte counts, analysis of the hematological parameters of the mice showed no significant differences between the experimental groups (Table 2). In the Fer.cream_AG8 and Fer.cream_AG9 groups, the amount of leukoytes increased, the increase being due to an increase in the absolute amount of lymphocytes. The relative proportion of lymphocytes in this group was also higher than in the control. Thrombocrit levels decreased in the same groups of animals. However, it should be noted that the hematological indices did not exceed the norms for mice.

Fer.cream_AG8 and Fer.cream_AG9 

Biochemical Indices

When analyzing biochemical parameters, special attention was paid to lipid metabolism and liver health parameters, as the animals were fed a product with a high fat content of 15 %. The HDL index changed in the experimental groups compared to the control group. In particular, it increased in the sour cream group and decreased in the fermented cream AG8 and AG9 groups (Fig. 1A). The LDLP index increased in all experimental groups compared to the control. The very low density lipoprotein index in Fer.cream_AG8 and Fer.cream_AG9 groups was significantly lower than control.

Figure 1: Biochemical indices of lipid metabolism in rat serum after 21 days of experiment: A – high density lipoproteins (HDLP), low density lipoproteins (LDLP), very low density lipoproteins (VLDL); B – triglycerides (TG), cholesterol (Chol). Click here to view Figure

Table 2: Effect of sour cream on hematologic indices of blood of mice after 21 days of experiment. (mean values ± SD, n = 7).

 ^a The results of the statistical analysis indicate significant differences between the groups at p < 0.05.

The amount of triglycerides in the blood of animals in the Fer.cream_AG8 and Fer.cream_AG9 groups was lower than in the control and sour cream groups. It should be noted that the lowest cholesterol level was observed in the group that received fermented cream of strain AG8. (Fig. 1B).

Figure 2 shows the activities in blood of the liver enzymes ALT and AST. It was found that ALT activity was significantly decreased in the group with fermented cream AG9 compared with the control. In case of AST enzyme, a decrease in its activity was observed in the group with fermented cream L.fermentum AG8, although there was a tendency to decrease AST activity in the group with sour cream AG9.

Figure 2: Changes in the activity of liver enzymes in the serum of mice treated with sour cream with different LAB.  Click here to view Figure

The addition of sour cream fermented by strains L.plantarum AG9 or L.fermentum AG8 to the diet slightly decreased the amount of total protein in the blood, but this indicator remained normal, although it differed from the control (Figure 3). When classical sour cream with L. cremoris and L. diacetilactis (F_cream group) was added to the diet, the amount of protein in the blood of mice increased slightly. Regular consumption of sour cream, regardless of the starter bacteria, led to an increase in bilirubin levels, but not above the normal level. It is important to note that the highest bilirubin elevation was in the classic sour cream group (F_cream group), while this index was lower in the groups with sour cream based on probiotic strains L.fermentum AG8 or L.plantarum AG9.

Figure 3: Variation in the amount of total protein and bilirubin in the serum of mice treated with sour cream with different LAB.  Click here to view Figure

Shifts in the abundance of a number of groups were observed when the gut microbial community was analyzed using classical microbiological methods (Fig. 4). Consumption of sour cream decreased the total number of heterotrophic facultative anaerobic and aerobic microorganisms in all experimental groups (Total bacteria). As for the LABs, their number increased in the F_cream group compared to the control and decreased in the Fer.cream_AG8 and Fer.cream_AG9 experimental groups. Unfortunately, it should be noted that the presence of sour cream in the daily diet led to an increase in the number of coliforms. However, in the variants with sour cream L.plantarum AG9 and especially L.fermentum AG8, the increase in the abundance of this group compared to the control was less intense. The comparable trend was found with the yeast population.

Figure 4: Changes in the number of different groups of microorganisms in the feces of mice treated with sour cream with different LAB.  Click here to view Figure

PCA and Correlation

PCA was utilized to investigate the associations between groups of mice fed diets fortified with sour cream and various starters (Fig. 5A). PCA was performed on two principal components, selected according to established criteria. The first principal component (PC1; X axis) had an explanation of 21.31% of the total variation in the data set, while the second principal component (PC2; Y axis) had an explanation of 15.29% of the total variation. The PCA values categorised the groups of mice into clusters. These clusters practically do not overlap. Their position in the coordinate system clearly indicates the essential role of the bacteria present in the sour cream. Depending on whether probiotic bacteria L.plantarum AG9 or L.fermentum AG8 were used instead of the classical starter, it is evident that the functional parameters of the mice were significantly improved.

Figure 5: Principal Component Analysis (A) of mouse group characteristics after 21 days of experiment and correlation (B) between blood parameters. Click here to view Figure

It is clear from the correlation analysis of hematological and biochemical parameters (Figure 5B) that there is a strong negative correlation. (a) Leucocytes, lymphocytes (WBC, LYM/TG, Chol, VLDL (TG, Chol, VLDL); (b) Erythrocyte population breadth/TG, Chol, VLDL, HDLC (TG, Chol, VLDL, HDLC); (c) AST/percentage of lymphocytes (LY %). Positive correlations were found only in similar groups of parameters.

Discussion

It is well known that lactic acid bacteria and products based on them have beneficial effects on macroorganisms, including humans.³⁰ In this work, we demonstrated the probiotic potential of lactic acid bacteria at the level of fermented cream under in vivo conditions in a mouse model. To study the physiological effects of sour cream fermented with different isolates on mice, we evaluated changes in body weight, organ indices, lipid profile, liver enzymes and haematological markers.

Mice that were fed a diet comprising sour cream fermented with L. fermentum AG8 and L. plantarum AG9 exhibited a modest yet persistent decline in body mass in comparison to the control group. Haematological data showed an increase in leukocyte and lymphocyte counts, particularly in the L. fermentum AG8 group (p < 0.05), which may indicate an enhanced immune response. Many studies have reported the positive role of lactobacilli in forming immune status.^31,32

Despite the diet including fatty sour cream, this decrease may indicate improved lipid utilisation or reduced fat accumulation. Analysis of organ indices revealed a significant decrease in liver and kidney weights in the AG8 and AG9 groups (p < 0.05), suggesting a possible reduction in metabolic burden. However, the spleen index increased in these groups, correlating with increased immune activity.

Introducing sour cream containing the L. fermentum AG8 and L. plantarum AG9 strains into the diet resulted in significantly lower triglyceride and total cholesterol levels than the control group and the group receiving classic sour cream containing L. cremoris and L. diacetilactis. Notably, mice receiving sour cream containing L. fermentum AG8 exhibited the lowest cholesterol levels (p < 0.05). While HDL levels increased in the classic sour cream group, they remained lower in the L. fermentum AG8 and L. plantarum groups. This suggests that these strains may regulate lipid transport and metabolism differently, possibly through the activity of bile salt hydrolases or by modulating the gut microbiota. In addition, there was evidence of the positive role of L. fermentum AG8 and L. plantarum AG9 strains in the regulation of mouse liver function in sour cream composition, as indicated by a significant decrease in the levels of ALT and AST activity.

The results, which included a reduction in triglycerides, cholesterol, bilirubin and liver enzymes, clearly indicate the feasibility of incorporating them into a high-fat product without compromising their probiotic functionality. These findings are particularly significant in the context of heightened interest in the use of non-fermented strains as probiotics, with the potential to enhance human health. There is an increasing focus on reducing elevated levels of lipids, low-density lipoproteins and triglycerides in the blood through dietary foods.

Some people may have a mild form of hypercholesterolaemia in which taking pharmaceuticals like statins is not justified given their side effects.³³ Probiotics, defined as “live microorganisms that, when administered in sufficient quantity, provide health benefits to the host”.³⁴ Probiotic LAB can be effective in lowering cholesterol levels even with a high-fat diet,^35-37 these probiotics are used to ferment milk.³⁸ Many authors studying Lactiplantibacillus plantarum have noted its ability to lower cholesterol, regulate gut flora and boost the immune system.^39-41 Limosilactobacillus fermentum strains have now been identified as the best candidates for use as probiotic lactic acid bacteria (LAB) with hypocholesterolemic effects.^38,42-44 In the present studies, the observed effect of reducing cholesterol and triglyceride levels when consuming sour cream fermented with Limosilactobacillus fermentum AG8 and Lactiplantibacillus plantarum AG9 may be due to the ability of these strains and their metabolites, particularly exopolysaccharides, to exert the lipase-inhibiting effect previously shown.²⁸

It appears that the microbiota in the gut may play a role in the regulation of cholesterol levels and the body’s immune system.^45-47 The addition of Lactococcus-based 15% fat sour cream to the diet of mice resulted in increased levels of coliform bacteria (e.g. Escherichia, Enterobacter, Klebsiella, Citrobacter) compared to the control, which was induced by an increase in the fat content of the diet. However, this increase was lower in mice receiving L.plantarum AG9 and especially AG8-based sour cream, which is a positive outcome. The observed effects of L.fermentum AG8 and L.plantarum AG9 sour cream are probably due to the antibacterial properties of the strains. This has been shown previously.^25,29

The present study definitively shows that the use of classic sour cream did not affect the leucocyte count. The control group and the F_cream group both had a lower content of leucocytes and lymphocytes, which indicates a reduced immunological status. Whereas the presence of the product with L.fermentum AG8 and L.plantarum AG9 in the diet of mice increased the number of leucocytes and lymphocytes to normal. The increase in the leukocyte level within the normal range may indicate the stimulation of immunity, which correlates with the increase in the spleen index. Meta-analyses of data from many researchers clearly show that strains of Lactobacillus plantarum species can significantly affect the immune system⁴⁸ and lipid profile.⁴⁹ Researchers have noted the positive role of L. fermentum in promoting mammalian health, including through changes in the composition of the microbiome.^50,51

In addition to the previously mentioned positive changes in the lipid profile and liver function indices, it is important to emphasize that the utilization of L. plantarum AG9 and L. fermentum AG8 in fermented sour cream may contribute to the improvement of the gut microbiota. This is because the gut microbiota plays a key role in lipid metabolism and immune homeostasis. It is hypothesized that alterations in the composition of intestinal flora may enhance gut barrier function, thereby reducing systemic inflammation and promoting metabolic normalization. Furthermore, exopolysaccharides, the production of which is characteristic of these strains, are likely to have a prebiotic effect, thereby stimulating the growth of beneficial bacteria and further supporting macro-organismal health.^26,28 The long-term utilization of probiotics has the potential to prevent metabolic disorders, particularly in individuals with a genetic predisposition to hypercholesterolemia, thereby reducing the necessity for pharmacotherapy, which is associated with significant adverse effects.

Our in vivo studies definitively demonstrate that LAB strains isolated from silage are promising probiotics for use in functional foods. These strains can effectively modulate lipid metabolism and reduce cholesterol levels, enhance immune status and correct gut microbiota. Notably, we observe these effects in high-fat foods, highlighting their significance. In summary, the integration of specific probiotic strains into high-fat foods offers new opportunities for the rational and safe management of lipid metabolism and immune enhancement.

Conclusion

This study definitively investigated the effect of classical 15 % sour cream based on Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetilactis, as well as sour cream fermented with probiotic Limosilactobacillus fermentum AG8 and Lactiplantibacillus plantarum AG9 on physiological and biochemical parameters of mice. The results show that there was a significant improvement in lipid metabolism, immune response and liver condition when sour cream fermented with these probiotic strains was included in the diet. Specifically, the consumption of sour cream fermented with L. plantarum AG9 and L. fermentum AG8 resulted in a decrease in triglycerides, cholesterol and liver enzymes (ALT and AST), indicating a potential protective effect against metabolic disorders induced by the high-fat product. Furthermore, an increase in the number of leukocytes and lymphocytes, as well as an increase in the spleen index, indicates an immunomodulatory effect of these probiotic strains.

The results clearly show that probiotic bacteria can be used in fermented dairy products to improve their functional properties without compromising traditional characteristics. There is no question that these probiotics promote cardiovascular health, as evidenced by the reduction in harmful lipid fractions (LDLP and VLDL) and the increase in beneficial HDLP levels. What is certain is that the changes observed in the composition of the gut microbiota, particularly the reduction in coliform bacteria, highlight the potential of these strains to improve gut health.

This study confirms that sour cream fermented with L. plantarum AG9 or L. fermentum AG8 is a functional food with significant health benefits, particularly for modulating lipid metabolism and enhancing immune function. These results are consistent with the growing interest in probiotics as a natural approach to treat metabolic and immune disorders. Further studies are needed to investigate the long-term effects and potency of these probiotic strains in the human diet, especially in the context of high-fat dairy products.

Acknowledgement

The Author would like to thank Kazan National Research Technological University and Kazan Federal University for the support.

Funding Sources

The work was funded by the grant of the Academy of Sciences of the Republic of Tatarstan, provided to young candidates of sciences (postdoctoral fellows) for the purpose of de-fending doctoral dissertation, performing research work, as well as performing labor functions in scientific and educational organizations of the Republic of Tatarstan within the framework of the State Program of the Republic of Tatarstan “Scientific and Technological Development of the Re-public of Tatarstan”, grant No. 117/2024-PD.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Kazan Federal University (protocol No. 40 of March 9, 2023).

Informed Consent Statement

This study did not involve human participants, animals or any such material and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to Reproduce Material from Other Sources

Not applicable.

Author Contributions

The sole author was responsible for the conceptualization, methodology, data collection, analysis, writing, and final approval of the manuscript.

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Abbreviations List

ALT – Alanine aminotransferase

AST – Aspartate aminotransferase

HDLP – High-density lipoprotein

LAB – Lactic acid bacteria

LDLP – Low-density lipoprotein

LY – Lymphocytes

LY % – Percentage of lymphocytes

PCA – Principal Component Analysis

TC – Total cholesterol

TG – Triglycerides