Effect of cultured white soft cheese on the histopathological changes in the kidneys and liver of albino rats

Table 1 shows the data on the chemical composition of white soft cheese made with different strains of L. helveticus, L. rhamnosus and S. thermophilus S₃₈₅₅ during pickling periods, which was stored in a refrigerator for up to 28 days.

pH values

The data in Table 1 indicated the significant difference in the pH values of white soft cheese (P < 0.05) among all cheese treatments. The pH values of the control cheese, L. helveticus cheese, L. rhamnosus cheese and S. thermophilus S₃₈₅₅ cheese ranged from 6.43 ± 0.03 to 5.21 ± 0.01, 6.46 ± 0.01 to 5.19 ± 0.00, 6.49 ± 0.02 to 5.08 ± 0.01 and 6.47 ± 0.01 to 5.28 ± 0.01, respectively. The pH values of all cheese samples gradually decreased during the storage peroid of up to 28 days because of the accumulation of lactic acid produced by the lactic acid bacteria. These results are in consistent those reported by Perotti et al.²⁵_, Mansour and Zaki²⁶ and Zonoubi and Goli²⁷.

Titratable acidity (TA %)

The data in the same table indicated that the acidity of white soft cheese increased with the increase in storage periods at refrigerator temperatures up to 28 days. The control samples had lower TA than those of the other treatments at the end of the pickling period (28 days) as the control samples doesn’t inoculated with any strain. Conversely, the treatment containing the L. rhamnosus strain had the highest TA% at the end of the storage period. The differences TA titratable acidity among the cheese treatments might be attributed to the varying growth rates of microorganisms and their ability to ferment lactose during the pickling period. The same trend was obtained by Mansour and Zaki²⁶, Zonoubi and Goli²⁷ and Naeim²⁸_.

Moisture content

The data in Table 1 indicated that the moisture content of white soft cheese increaed with the increase in pickling periods at refrigerator temperatures up to 28 days. The treatment containing the L. helveticus strain and stored for 28 days had a higher moisture content than the other treatments. This increase of moisture content could be attributed to the refrigerator temperature which helps to imbibe the whey into the curd. Similar results were reported by Kebary et al.⁵.

Protein content

White soft cheese showed a clearly significant (P < 0.05) decrease in protein content during the pickling period in all treatments. The decrease in protein content related to the increase in the moisture content of white soft cheese during the pickling period. However, the protein content of cheese samples was agreed with the results that found by^29,30,31.

Fat/dry matter (F/DM)

The data in the same table indicated that, the F/DM of white soft cheese increased with the increase in pickling periods at refrigerator temperatures up to 28 days in all treatments. Moreover, the cheese samples containing the S. thermophilus S₃₈₅₅ strain had a lower F/DM than the other cheese tsamples at at the end of the pickling period (28 days). Conversely, the treatment containing the L. helveticus strain had the highest F/DM at the end of the pickling period. Similar results were obtained by Kebary et al.⁵ and El-Sayed and El-Sayed³¹.

Table 2 shows the microbiological analyses of white soft cheese made with deferent strains of lactic acid bacteria (L. helveticus, L. rhamnosus and S. thermophilus S₃₈₅₅) and pickled at refrigerator temperatures up to 28 days.

Streptococci counts

Significant differences (P < 0.05) in streptococci count (Tale 2) were observed, and the count ranged from 3.65 ± 0.03 to 8.67 ± 0.01 log cfu/g^-1. Moreover, it was indicated that the control samples had lower streptococci counts than other cheese samples. Conversely, the samples of S. thermophilus S₃₈₅₅ cheese had the highest streptococci counts (8.67 ± 0.01 log cfu g^-1) at 7 days of the pickling period. The streptococci counts of L. helveticus cheese and L. rhamnosus cheese were higher than that of the control cheese. This may be related to the synergistic effect between lactobacilli and streptococci during ripening. Moreover, the streptococci counts of were increased in the beginning days during the storage period and then decreased up to the end of pickling period. These results are consistent with those obtained by Papademas et al.³² and Dafalla and Abdel Razig³³.

Lactobacilli count

The obtained data in the same table indicated that the treatment of L. helveticus cheese and L. rhamnosus cheese had higher lactobacilli counts than the control and S. thermophilus S₃₈₅₅ cheese. Moreover, the lactobacilli counts were increased with the increase in storage periods to reach the highest count in 14 days of the pickling period in all cheeses. For the except S₃₈₅₅ cheese, if the highest count was reached in 7 days, then the counts were decreased in all treatments up to the end of the pickling period. Moreover, the data of lactobacilli counts indicated significant differences (P < 0.05) among all treatments, the count ranging from 3.70 ± 0.10 to 8.67 ± 0.01 log cfu g^−134,35,36_.

Total bacterial count

Table 2 showed significant differences (P < 0.05) and indicated that the control samples had lower total bacterial counts than the other cheeses. Moreover, the highest counts were obtained from the S. thermophilus S₃₈₅₅ cheese samples that were pickled for 7 days. By contrast the lowest total counts were obtained from the fresh control samples. Furthermore, the total bacterial counts were increased at the beginning of the pickling period and then decrease up to 28 days in all treatments. These results are agreed with those of Kebary et al.⁵ and Dafalla and Abdel Razig³³.

Figure 1 shows the bacteriological properties (streptococci count, lactobacilli count, total bacterial count and coliform count) of rat feces before and after feeding with white soft cheese.

Streptococci count

The obtained data in Fig. 1A indicated that, the streptococci counts before and after feeding with white soft cheese did not show any significant difference (P > 0.5) . In all rat groupd, it is clear that the streptococci counts after feeding with white soft cheese were higher than those before feeding due to the increase in the number of streptococci in inoculated and uninoculated cheese. These results are similar with those obtained by³⁷.

Lactobacilli count

The obtained data in Fig. 1B indicated that, the lactobacilli counts before and after feeding with white soft cheese among all rat groups showed significant difference (P < 0.05). Also, lactobacilli counts after feeding with white soft cheese were higher than those before feeding in all rat groups. Furthermore, Group 2 had the highest lactobacilli count before feeding with white soft cheese, whereas Group 3 had the highest lactobacilli counts after feeding.

Total bacterial count

The obtained data in Fig. 1C showed significant differences (P < 0.05) and indicated that the treatment of Group 1 initialy had lower total bacterial count than that of the other expermintal groups, whereas , the treatment of Group 4 had the highest count. Conversly, the treatment of Group 1 after feeding had lower total bacterial counts than that of the other treatments whereas the treatment of Group 3 had the highest count.

Coliform count

The obtained data in Fig. 1D indicated that the in treatment Group 3 initialy had the lowest coliform counts than that of the other treatments, whereas the treatment of Group 1 had the highest counts. Conversely, the treatment of Group 4 after 4 weeks of feeding had the lowest counts of coliform groups than that of the other treatments, whereas the treatment of Group 1 had the highest count. The data also indicated that the coliform count decreased after the feeding period in all groups, which could be attributed to the presence of lactic acid bacteria.These results are in agreement with those obtained by³⁸, who found that the probiotic strain L. plantarum as a single species or in combination with oil (Lini oleum virginale) decreased the total coliform count of and increased the lactobacilli in the feces of rats.

Histopathological observations

Renal histopathological changes

The histopathological changes in the liver tissue have been observed in all experimental groups apart from the control rats on the basis of the typical histological architecture of the normal renal parenchyma (Fig. 2) as observed previously by Brzóska et al.³⁹.

However, the sections taken from the rat kidneys of Group II (treated with basal diet 70% + control cheese 30%) showed several pathological findings: the cortical area showed glomerular atrophy, and the glomerular tuft was condensed and BS appeared to be wider than normal (Fig. 3A–C). Proximal and distal convoluted tubules exhibited degenerated epithelial lining. The vasculatures at the cortical area showed severe congestion; the blood vessels were dilated widely and engorged with blood (Fig. 3B,C). The tubules in the renal medulla showed vacuolation, degeneration and desquamation of its lining epithelium (Fig. 3D), and intense congestion was oserved in the blood vessels between the renal tubules (Fig. 3D).

The renal sections of Group III rats (treated with basal diet 70% + cheese containning L. helveticus 30%) showed glomerular atrophy, and some renal corpuscles revealed a variable degree of glomerular loss (Figs. 4A,B). Degeneration of proximal and distal convoluted tubules was observed and the epithelium was desquamated inside the lumen of some tubules and appeared as epithelial casts (Fig. 4B,C). The arcuate artery located at the corticomedullary junction was dilated and congested with thickening of its wall due to fibroses. Perivascular edema and hemorrhage around the arcuate artery (Fig. 4D,E). Henle’s loop showed dilatation with desquamated epithelium inside its lumen (Fig. 4F).

The histopathological examination of the kidneys of Group IV (treated with basal diet 70% + cheese containing L. rhamnosus 30%) showed that the changes of the glomeruli comprise atrophy, distortion, and hypocellularity of the glomerular tuft. BS was widened and the nucleus of the remaining capillary tuft was condensed (Fig. 5A). The renal tubular epithelium showed a series of degenerative changes up to necrosis; both proximal and distal convoluted tubules were affected. The tubular epithelium sometimes showed the lysis of the cytoplasm and pyknosis of the nucleus. Sometimes the necrotic epithelium sloughed and the tubules were dilated. Changes in the tubules were diffuse and involved a massive area of the both cortex (Fig. 5B) and medulla (Fig. 5F). The most prominent histopathologic changes were present mainly in the renal vasculature of the glomeruli and renal tubules. The primary changes were present in the renal vasculature, especially those in the cortical area (Fig. 5C). The interstitial capillaries were dilated and engorged with blood. In some cases, the blood vessels at the corticomedullary junction were congested with a very mild perivascular edema (Fig. 5D,E). Focal small and multiple areas of lymphoid cell reactions were oserved in the interstitial areas and around the vasculatures (Fig. 5D,E). Our results are consistent with those of Al-Mathkhury and Baraaj⁴⁰ who found that the renal tissues of the rats that were injected intraperitoneally with Lactobacillus bulgaricus, Lactobacillu plantarum and Lactobacillu acidophilus showed congestion of the vessels with blood and hemorrhage.

The kidneys of Group V (treated with basal diet 70% + cheese containing S. thermophilus S₃₈₅₅ 30%) showed prominently glomerular atrophy and hypocellularity, which were associated with the widening the BS (Fig. 6A). Sever cortical vascular congestion (Fig. 6B), Severe necrotic changes of renal tubular epithelium in both cortex (Fig. 6C) and medulla (Fig. 6D); the necrotic changes were severe and diffuse , which resulted in the loss of the entire epithelium. Hemorrhage at the corticomedullary junction (Fig. 6C). The intertubular blood vessels were congested (Fig. 6D).

The kidneys of rats that were fed with Domiati cheese stored in a refrigerator exhibited regions of hemorrhage and edema in the tubules and in the interstitial areas. Moreover, it showed the degeneration of renal tubules; and partly the degeneration of renal corpuscles^9,10.

Based on the histopathology scores, the severity of glomerular atrophy (Fig. 7A), renal tubular degeneration (Fig. 7B) and vascular congestion (Fig. 7C) were significantly increased (P ≤ 0.05) in all treated experimental groups compared with the control Group.

Hepatic histopathological changes

The microscopic examination of the rat liver of Group I revealed normal hepatic architectures which comprised, normal central vein, hepatic cords, hepatocytes and portal area contents (bile duct, hepatic artery, and vein) (Fig. 8).

Liver sections taken from the rats kidneys of Group II (treated with basal diet 70% + control cheese 30%) showed variable hepatic injuries such asthickening in the Glisson capsule surrounding the liver with extensive hemorrhage and edema (Fig. 9A). Central vein dilated and engorged with blood (Fig. 9B), dissociation, and disorganization of hepatic cords, and vacuolation of hepatocytes (Fig. 9C). The portal areas involved wide areas in hepatic parenchyma as the portal veins extensively were dilated and filled with blood with periportal edema mixed with fine threads of fibrosis which branched in-between hepatic tissue (Fig. 9D).

The liver sections of Group III (treated with basal diet 70% + cheese containing L. helveticus 30%) showed congested central veins with degeneration and desquamation of its endothelium (Fig. 10A). Hepatocellular vacuolar degeneration, hepatic cells appearing as empty spaces with a marginally located flattened nucleus which similar to the appearance of signet rings (Fig. 10B). The portal areas showed congestionin the portal vein with severe periportal fibroses which extends as tracts between hepatic lobules (Fig. 10C,D).

The histopathological examination of the rat liver of Group IV(treated with basal diet 70% + cheese containg L. rhamnosus 30%) showed dilated central vein with degenerated endothelial lining (Fig. 11A) and vacuolar degeneration of hepatocytes (steatotic like cells) (Fig. 11B,C). The presence of focal areas of nodular hyperplasia, the hyperplastic cells mixed with lymphocytic infiltration (Fig. 11D,E), other focal nodular areas exhibiting stellate scar with abnormal blood vessels, the vessels showing a marked thickening of the wall without lumen (Fig. 11A,F). Wide portal areas due to extensive widening and congestion in the portal veins with periportal fibrosis and edema, and the presence of newly formed nonfunctional bile ductulus (Fig. 11G–I). Similar results were obtained by³⁹ who found that the liver tissues of the rats that were injected intraperitoneally with L. bulgaricus, L. plantarum and L. acidophilus showed inflammatory cells infiltration and also necrosis.

The liver sections of Group V (basal diet 70% + cheese contaning S. thermophilus S₃₈₅₅ 30%) showing congested central vein (Fig. 12A), and vacuolation in hepatocytes (Fig. 12B). The focal areas of fibrosis with mild mononuclear lymphocytic infiltration (Fig. 12C). The focal areas of coagulative necrosis were also found (Fig. 12D). Wide portal areas showed extensively wide portal veins filled with blood with severe periportal edema (Fig. 12E,F).

The liver of rats fed with Domiati cheese stored in a refrigerator revealed vacuolation in the cytoplasm of the hepatocytes. Otherwise, the liver of the other rats showed the normal structure of the hepatic lobules. The histopathological changes that happened in the liver of rats were perhaps due to an increase in the levels of the biogenic amines of Domiati cheese^9,10.

Based on histopathology scoring, the severity of vascular congestion (Fig. 13A), hepatocellular changes (Fig. 13B) and periportal fibrosis (Fig. 13C) were significantly increased (P ≤ 0.05) in all treated experimental groups compared with the control Group.

Probiotics may theoretically be implicated in inducing different side effects as:

Systemic infections, excessive immune stimulation in susceptible individuals, gene transfer and deleterious metabolic activities; those in according to a 2002 report jointly released by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United Nations (http://www.fda.gov/ohrms/dockets/dockets/95s0316/95s-0316-rpt0282-tab-03-ref-19-joint-faowhovol219.pdf).

Our experimental results showing several systemic disturbances in liver and kidney cellular metabolism which alter its morphology in experimental groups, in addition to several vascular and inflammatory changes. Our findings were in agreement with previously recorded studies, which found many side effects after probiotic administration^{41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62}.

Lactobacillus bacteremia and overt sepsis have previously been observed in association with S. boulardii [cerevisiae], Lactobacillus GG, Bacillus subtilis, Bifidobacterium breve, or combination probiotics^{41,42,43,44,45,46,47}. Endocarditis has also been observed as a result of both Lactobacillus and Streptococcus probiotics^48,49. Lactobacillus rhamnosus has also been linked to the formation of an abscess on two occasions^50,51.

Because probiotics have been found to alter both the innate and adaptive immune systems, including cytokine release and dendritic cell function^52,55, there has been some worry regarding the risk of overstimulating the immune system in some individuals, possibly leading to autoimmune phenomena or inflammation.

It was confirmed that, plasmids carrying genes giving resistance to tetracycline, erythromycin, chloramphenicol or lincosamide, macrolide, streptomycin, and streptogrammin are found in lactic acid bacteria^56,58. Transfer of conjugation from enterococci to lactobacilli and lactococci can occur in animals’ guts as well as in vitro; however, transfer to lactobacilli is uncommon ⁵⁹.

One clinical trial raised serious concerns about the safety of probiotics⁶⁰. The subjects in the probiotic arm of the trial had a greater mortality rate, which was ascribed to intestinal ischemia. The scientists speculated that probiotic bacteria delivery raised the oxygen demand in the gut mucosa, despite the fact that blood flow was already modest. Alternatively, the probiotics could have caused an inflammatory response in the small bowel, resulting in decreased capillary blood flow. In patients with pancreatitis, two previous smaller-scale investigations (later combined to boost power)^61,62 showed a reduction in septic sequelae, surgical intervention, and infected necrosis in patients with pancreatitis given a symbiotic containing lactic acid bacteria and fiber.

As a result, many investigators have engaged in discussions with regulators concerning the challenges these policies pose to those seeking to advance scientific knowledge regarding the efficacy and safety of these products. It is critical to validate the identity of the organism through molecular testing at a reference laboratory if an infection appears to be caused by a probiotic strain and this aspect will be taken in our consideration in the following research work.