Fermented milk supplemented with water-soluble curcumin ameliorates survival rate, selected biochemical parameters and fecal microbiota of Ehrlich ascites carcinoma-bearing mice

This study assessed the impact of fermented cow and soy milk supplemented with curcumin on enhancement the health status of female Swiss mice under Ehrlich ascites carcinoma (EAC) condition. Both types of milk were fermented by Lactobacillus plantarum EMCC 1027. Ninety-five grams of basal diet mixed with five grams of either fermented cow or soy milk supplemented with curcumin. Induction of Ehrlich tumor-bearing mice synergized by intraperitoneal injection with 1 × 106 Ehrlich ascites tumor cells after one week of the adaptation period. Our results showed that levels of survival rate were increased to 117% in mice that were fed fermented cow and soy milk while supplementation of fermented soy milk with curcumin improved the survival rate to 150%. The concentration of glutathione and superoxide dismutase in Ehrlich tumor-bearing was increased after feeding with both types of fermented milk. Fermented soy milk supplemented with curcumin showed the highest reduction levels of plasma tumor necrosis factor (TNF)-α and interleukin-6. The relative gene expression TNF-α was also significantly (P < 0.05) down-regulated in mice that were fed fermented soy milk with curcumin. The viable count of lactobacilli and bifidobacteria in feces samples was increased when mice fed both types of fermented milk. Dietary feeding with fermented cow or soy milk supplemented with curcumin has an ameliorative impact on the survival rate, antioxidant capacity, inflammation, fecal microbiota in EAC mouse model. Further research is still needed to investigate the molecular mechanisms in this tumor. Fermented cow or soy milk- curcumin formula enhances the survival rate of EAC mice. It enhances antioxidant capacity and lowers pro-inflammatory cytokines. It increases fecal lactobacilli and bifidobacteria. Fermented cow or soy milk- curcumin formula enhances the survival rate of EAC mice. It enhances antioxidant capacity and lowers pro-inflammatory cytokines. It increases fecal lactobacilli and bifidobacteria.


Introduction
Generally, the global occurrence of cancer has increased.In 2018, about 9.6 million cancer-related deaths were recorded [1].In Egypt, Carcinoma of the breast is the most prevalent cancer among Egyptian women representing 29% of National Cancer Institute cases [2].The occurrence of cancer is due to several key factors such as unhealthy lifestyle, aging, and socio-economic development [3].
Probiotic fermented milk and soy milk had shown an inhibitory effect against different cancer models.In this context, it has been demonstrated that yogurt and soy yogurt containing bifidobacteria could inhibit the proliferation of Ehrlich ascites tumor cells [4].Different probiotic strains had anticarcinogenic activities [5,6].Also, yogurt manufactured by Lactobacillus (L.) helveticus R389 suppressed tumor growth and modulated the inflammatory system positively [7].Furthermore, Kefir grains could inhibit Ehrlich ascites tumor cells through induction of apoptosis and immunomodulation [8].
Curcumin as a functional ingredient has a high content of polyphenols and has been used extensively in Ayurveda medicine for centuries.Curcumin, as a complementary medicine, has shown anti-carcinogenic activity through inhibition of different inflammatory pathways as well as the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway [9,10].Our previous results showed that fortification of probiotic fermented milk and soy milk with water-soluble curcumin had an enhancement effect on anti-carcinogenic activity using in vitro model [11].Therefore, the present study aimed at exploring the influence of probiotic fermented cow/ soy milk supplemented with curcumin, which proposed as formula for Egyptian market, on enhancement of the survival rate of Ehrlich ascites carcinoma (EAC) mice, improvement of the antioxidant status, reduction of the selected pro-inflammatory cytokines e.g.TNF-α and modulation of fecal microbiota.

Materials
Lactobacillus (L.) plantarum EMCC 1027 was obtained from the Egyptian Microbial Culture Collection, Cairo, Egypt.Water-soluble curcumin was purchased from DolCasTenshi Bioceuticals Co. (New Jersey, USA).All microbiological media were purchased from Sigma-Aldrich Co. (St.Louis, MO, USA).Ehrlich tumor cells were obtained from the National Cancer Institute, Cairo University.Female Swiss albino mice were purchased from Ophthalmology Institute, Giza, Egypt.

Propagation of L. plantarum cultures
L. plantarum EMCC 1027 was sub-cultured in de Man, Rogosa and Sharpe (MRS) broth medium and incubated aerobically at 37 °C for 18 h.

Preparation of fermented cow and soy milk supplemented with curcumin
Fermented cow and soy milk supplemented with watersoluble curcumin were prepared as previously described by 11 .Briefly, Soy milk or cow milk (total solids 12%) was inoculated with freeze-dried L. plantarum culture at 0.02% and incubated at 37 °C till the pH of final product was reached to 5.0 then curcumin was added at 150 mg/100 mL for both types of milk.

Ethics statement
All animal procedures were approved by Cairo University-Faculty of Science, Institutional Animal Care and Use Committee (IACUC) with approval Number: CU/I/F/29/21 and performed according to the Guide of animal care and use.

Animals and feeding protocol
The female Swiss albino mice (weight: 20-23 g, age: two months old) were maintained in an air-conditioned room at a temperature of 22 ± 1 °C and humidity of 55 ± 1% with a 12 h light/dark cycle and had ad libitum access to water.All animals were kept under normal healthy conditions and fed a basal diet (MILAD Co., Nasr city, Cairo, Egypt) for one week.After this adaptation period, all mice were divided randomly into six experimental groups each of 10 animals as the following: Group 1 (Normal group): mice were fed a basal diet.The composition of the basal diet was as previously described by [12].Group 2 (Control group): mice were intraperitoneally (IP) injected with 1 × 10

Blood collection from survived mice
At the end of the experiment (after twenty days), the survival percentage in each group was calculated as follows: survival percentage = (number of survived animals/total number of animals) × 100.Mice were lightly anesthetized by isoflurane.Blood samples were collected from the retinal orbital plexus through heparinized capillary tubes into heparinized tubes, separated by centrifugation (3500×g for 15 min), and stored at − 20 °C until analysis.

Determination of biochemical parameters and cytokine levels
Plasma glutathione and superoxide dismutase (Thermo Fisher Scientific, Passau, Germany) were determined using a spectrophotometer (SHIMADZU, Tokyo, Japan).Interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured by commercial ELISA kits (Genzyme Diagnostics, Cambridge, England) according to manufactured instructions.Optical density was measured at 450 nm.

Detection of TNF-alpha relative gene expression
The detection of the TNF-α gene in liver tissue by reverse transcription-polymerase chain reaction (RT-PCR) was performed as described previously by [8,13].

Enumeration of fecal lactobacilli, bifidobacteria, and Enterobacteriaceae
A specimen of 0.1 g of feces from each mouse was freshly collected at the end of the experiment and transferred into the anaerobic tube (Clinical Supply, Gifu, Japan).Samples were examined for total lactobacilli using MRS agar medium pH 5.3 (37 °C/72 h), bifidobacteria using TOS-MUP agar medium (37 °C/72 h, anaerobically), and total Enterobacteriaceae crystal using violet agar (Merck, Darmstadt, Germany, 37 °C/24 h) [14].

Statistical analysis
Results were statistically analyzed with the SAS statics package (version 9.4, SAS Institute, Cary, NC).One-way ANOVA was used to assess the significance of differences between groups with P < 0.05 being considered significant.Data were expressed as mean ± standard deviation (SD).Duncan test and one-way analysis of variance were used.

Survival rate of mice
The present study assumed that fermented cow/soy milk supplemented with curcumin has therapeutic effect against breast cancer in mice model.A comparison between fermented cow milk and another one of plant origin i.e. soy milk was assessed.Additionally, the protective impact of supplementation of both types of fermented milk with curcumin was investigated.
The mean survival rate of Ehrlich tumor-bearing mice was 60%.Feeding basal diet either with fermented cow or soy milk significantly (P < 0.05) increased the survival rate of Ehrlich tumor-bearing mice to 117%.Fermented cow milk supplemented with curcumin diet caused an increase in the survival rate to 133% while feeding mice with fermented soy milk supplemented with curcumin resulted in an increase in the survival rate to 150% (Table 1).This survival rate was higher than non-fermented soy milk reaching 108% [15].Thus, the process of fermentation presented a more beneficial impact than the unfermented form.Moreover, the antitumor activity of fermented milk against HT-29 and Caco-2 cells has been documented through suppression of cell proliferation and reduction of lung metastasis by supplementation of fermented milk [16].The progression of breast tumors was suppressed in the rats fed a fermented soymilk diet [17] due to the antioxidant activity of fermented milk [18].Curcumin is one of these phytochemicals that has received a lot of attention due to its effectiveness against a variety of cancer types.It has antioxidant, anti-inflammatory, and cancer cells antiproliferative activity against cancer cells [19].In the present study, the use of formulated water-soluble curcumin resolved the previous poor water solubility of curcumin.https://doi.org/10.1007/s42452-023-05493-7 Providing diet pellets with soluble curcumin enhanced the higher survival rate of Ehlrich ascites-bearing mice to 133% in the case of fermented cow milk and 150% in the case of fermented cow milk.This result agreed with previous studies that curcumin could prevent colorectal cancer proliferation [20,21] and enhance the survival rate of Ehrlich ascites carcinoma mice [22].

Levels of glutathione and superoxide dismutase
The plasma levels of SOD and GSH in the Ehrlich tumorbearing group showed a significant decrease compared to the normal group.All other treatments induced significantly higher levels (P < 0.05) of SOD and GSH compared to the Ehrlich tumor-bearing group (Fig. 1).Fermentation is an effective strategy for enhancing the supply of natural antioxidants because it boosts antioxidative activity by increasing the release of phenolic compounds from plant-based diets.Antioxidant activity in soy milk was increased after fermentation [23].As a result of many metabolic activities, reactive oxygen species (ROS) are continuously created that cause harm to DNA, proteins, lipids, and carbohydrates, contributing to multiple diseases such as cancer, atherosclerosis, and arthritis.Antioxidants serve a critical function in scavenging ROS.SOD and GSH performed an important antioxidant defense that converts superoxide radicals into molecular oxygen and hydrogen peroxide, protecting tissues from oxidative damage.The Ehrlich tumor-bearing mice that were supplemented with fermented soy milk exhibited higher levels of both SOD and GSH than those supplemented with fermented cow milk.This finding is matched with previous research that found soya milk has a much stronger antioxidant activity than cow milk [24].The same finding of higher levels of antioxidant enzymes was also observed in the Ehrlich tumor-bearing mice supplemented with fermented cow or soy milk plus curcumin.According to the study by [25], the phenol content of turmeric-fortified soya milk was higher than that of turmeric-fortified cow milk.

Plasma levels of IL-6 and TNF-α
As demonstrated in Fig. 2, the mean level of IL-6 was significantly (P < 0.

Gene expression of TNF-α
On the gene level, the relative gene expression of TNF-α was (8.51 ± 1.  3).TNF-α, interferon (IFN)-γ, and interleukin (IL)-6 are among the pro-inflammatory mediators produced during inflammation into blood stream causing hepatic inflammation and fibrosis, immune system failure and the excessive inflammatory reaction has related to cancer development [26].Phenolic compounds are one of the important bioactive components which act as natural antioxidants and immunological modulators that are created during [27].Dietary regulation of excessive inflammation is crucial for maintaining health.The elevated plasma levels of IL-6 and TNF-α in Ehlrich tumor-bearing mice were decreased by supplemented diet with fermented cow and soy milk.Also, the gene expression of TNF-α was down-regulated after both treatments.Our results in Figs. 2 and 3 are in accordance with a previous study reported that fermented milk contains bioactive peptides that stimulate lymphocyte proliferation and control the generation of pro-inflammatory cytokines such as IFN-γ and TNF-α in Balb/c mice [28].Furthermore, feeding probiotic L. reuteri ATCC 6475 could reduce levels of IL-6 in mice [29].Additional animal study confirmed our results whereas oral administration of L. acidophilus had anti-breast cancer activity in mice models [30].Finally, our results show a good correlation between supplementation of fermented cow/soy milk with curcumin and improving the antioxidant status of mice.

Fecal examination
The total viable count of fecal Enterobacteriaceae, lactobacilli, and bifidobacteria was illustrated in Enterobacteriaceae as a member of the gut microbiome plays a vital role in the development of breast cancer through increased secretion of β-glucuronidase [31,32].Our results in Table 2 showed a significant increase in the viable count of total Enterobacteriaceae in the Ehrlich tumor-bearing group.On the other hand, feeding mice with fermented cow milk or soy milk with /without curcumin lowered levels of Enterobacteriaceae and increased viable counts of lactobacilli and bifidobacteria in faces samples.The modulation of fecal microflora in mice may be due to the synergistic effect of both types of fermented milk and curcumin through their antimicrobial activities and prebiotic e.g.raffinose oligosaccharides content in soy milk [11].Our results are in agreement with results obtained by Feng et al. [33] whereas feeding fermented pasteurized milk could reduce levels of pro-inflammatory TNF-α and IL-6 level and could restore the gut microbiota in rat model.

Conclusion
Different in vitro or in vivo model was performed for evaluating the health benefits of probiotic strains [34].Furthermore, several ingredients e.g.grape seed extracts, curcumin and non-viable yeasts were added to different types of fermented milk in order to enhance their therapeutic effects [35,[35][36][37].This investigation found that formula consists of water soluble curcumin plus fermented either cow or soy milk by L. plantarum had an had an improvement impact on health condition of Ehrlich ascites carcinoma mice model by increasing the survival rate and the antioxidant capacity.Furthermore, it could reduce levels of plasma pro-inflammatory mediators (TNF-α and IL-6) and relative gene expression of TNF-α.The feeding diet positively modulate the fecal microbiota whereas the viable count of fecal lactobacilli and bifidobacteria increased in feces samples of mice treated with fermented milk mixed with curcumin.Thereby, the obtained data highlighted the link between fermented milk plus curcumin and enhancing of antioxidant status, lowing pro-inflammatory mediators and restoring gut microbiota.Therefore, this study recommends that the supplementation of fermented cow/ soy milk with curcumin for enhancing the health status of EAC mice and improving the therapeutic effect of fermented milk.Indeed, extensive research on molecular mechanisms of fermented milk-curcumin formula is still needed.

Fig. 2 Fig. 3
Fig.2Plasma levels of IL-6 (pg/mL) and TNF-alpha (ng/mL) in all mice groups.Data were expressed as a mean ± standard deviation.Mean values marked with different superscript letters are significantly different (P < 0.05).Mice were fed a basal diet (Normal); mice were injected intraperitoneally with 1 × 10 6 Ehrlich ascites

Table 1
The survival rate of mice during 20 days of the experiment N: number of animals; mice were fed a basal diet (Normal); mice were injected intraperitoneally with 1 × 10 6 Ehrlich ascites tumor cells after the adaptation period (Ehrlich-tumor); Ehrlich-tumor bearing mice were fed 95 g basal diet mixed with 5 g fermented cow milk (Cow milk), 5 g fermented cow milk supplemented with 150 mg curcumin (Cow milk + cur); 5 g fermented soy milk (Soy milk), 5 g fermented soy milk supplemented with 150 mg curcumin (Cow milk + cur)

Table 2 .
The mean viable count of Enterobacteriaceae in the Ehrlich tumor-bearing group was significantly increased in comparison to the normal group.On the other hand, all other treated groups showed a lower count of Enterobacteriaceae compared to Ehrlich tumor-bearing group.The mean viable count of lactobacilli and bifidobacterial was significantly decreased in Ehrlich tumor-bearing group compared to the normal group.All other treatments significantly elevated the viable count of lactobacilli and bifidobacteria compared to Ehrlich tumor-bearing group.