Lipolytic Postbiotic from Lactobacillus Paracasei Manages Metabolic Syndrome in Albino Wistar Rats

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Article Lipolytic Postbiotic from Lactobacillus paracasei Manages Metabolic Syndrome in Albino Wistar Rats

Ali Osman 1 , Nashwa El-Gazzar 2 , Taghreed N. Almanaa 3, Abdalla El-Hadary 4 and Mahmoud Sitohy 1,*

1 Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt; [email protected] 2 Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt; [email protected] 3 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] 4 Biochemistry Department, Faculty of Agriculture, Benha University, Benha 13736, Egypt; [email protected] * Correspondence: [email protected]; Tel.: +20-106-527-2667

Abstract: The current study investigates the capacity of a lipolytic Lactobacillus paracasei postbiotic as a possible regulator for lipid metabolism by targeting metabolic syndrome as a possibly safer anti- obesity and Anti-dyslipidemia agent replacing atorvastatin (ATOR) and other drugs with proven or suspected health hazards. The high DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,20-azino-bis (3-ethyl benzothiazoline-6-sulphonic acid)] scavenging activity and high activities of antioxidant enzyme such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) of the Lactobacillus paracasei postbiotic (cell-free extract), coupled with considerable lipolytic activity, may support its action against metabolic syndrome. Lactobacillus paracasei isolate was obtained from an Egyptian cheese sample, identiﬁed and used for preparing the postbiotic. The postbiotic Citation: Osman, A.; El-Gazzar, N.; was characterized and administered to high-fat diet (HFD) albino rats (100 and 200 mg kg−1) for Almanaa, T.N.; El-Hadary, A.; Sitohy, nine weeks, as compared to atorvastatin (ATOR; 10 mg kg−1). The postbiotic could correct the M. Lipolytic Postbiotic from disruption in lipid metabolism and antioxidant enzymes in HFD rats more effectively than ATOR. Lactobacillus paracasei Manages The two levels of the postbiotic (100 and 200 mg kg−1) reduced total serum lipids by 29% and 34% Metabolic Syndrome in Albino Wistar Rats. Molecules 2021, 26, 472. and serum triglyceride by 32–45% of the positive control level, compared to only 25% and 35% in −1 https://doi.org/10.3390/molecules ATOR’s case, respectively. Both ATOR and the postbiotic (200 mg kg ) equally decreased total 26020472 serum cholesterol by about 40% and 39%, while equally raising HDL levels by 28% and 30% of the positive control. The postbiotic counteracted HFD-induced body weight increases more effectively Academic Editor: Béla Juhász than ATOR without affecting liver and kidney functions or liver histopathology, at the optimal dose Received: 4 December 2020 of each. The postbiotic is a safer substitute for ATOR in treating metabolic syndrome. Accepted: 13 January 2021 Published: 18 January 2021 Keywords: Lactobacillus paracasei; cell-free extract; dyslipidemia; hypercholesterolemia; obesity; metabolic syndrome; antioxidants activity; antioxidant enzymes Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 1. Introduction Hypercholesterolemia is one of the major risk factors contributing to CVD (cardiovascular disease), as an elevation amounting to 1% increase in plasma cholesterol is associated with a double elevation (2%) of CVD incidence. The latter is the leading cause of mortal- Copyright: © 2021 by the authors. ity and morbidity in humankind [1,2]. High cholesterol levels can be very damaging to Licensee MDPI, Basel, Switzerland. people’s health, causing various severe health problems [3]. Alternatively, hyperlipidemia, This article is an open access article a chronic metabolic disorder, is characterized by abnormalities in lipid and lipoprotein distributed under the terms and metabolism, triggering many complications, such as atherosclerosis and hypertension [4]. conditions of the Creative Commons High-fat diet (HFD)-induced hyperlipidemia is usually associated with high levels of Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ total cholesterol, triacylglycerols, and low-density lipoprotein (LDL), but a low level of 4.0/). high-density lipoprotein (HDL) [5]. Long-term hyperlipidemia may further deteriorate

Molecules 2021, 26, 472. https://doi.org/10.3390/molecules26020472 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 472 2 of 22

general health by inducing nonalcoholic fatty liver disease (NAFLD), which implies hepatic steatosis and liver damage [6]. Hyperlipidemia and obesity are positively associated and can lead to coronary heart disease [7,8]. They represent the main components of metabolic syndrome, defined as a state of disturbed metabolic homeostasis characterized by visceral obesity, atherogenic dyslipidemia, arterial hypertension, and insulin resistance [9]. Obesity is currently recognized as the most wide-spread medical and social problem, deteriorating the general human condition, working ability, and patients’ quality of life [10]. Obesity and dyslipidemia are being widely treated with different drugs, including atorvastatin [9] and some natural products [11]. Despite its efficacy and a broad spectrum of action in lowering both low-density lipoprotein cholesterol (LDL-C) and triglyceride levels [12], atorvastatin (ATOR) may have potentially negative effects on the hepatic function [13]. This may in turn necessitate the search for safer alternatives. Probiotic bacteria, prebiotic compounds, and native or modified proteins are health- friendly approaches to preserve food or enhance its biological value [14–28]. Probiotics are microorganisms beneficial to the host’s health when consumed in adequate amounts. They are not a direct source of material but rather producers of many bioactive substances with diversified activities, known generally as postbiotic [29–36]. To adequately perform their function, they need other stimulating or activating compounds known as prebiotics, which not only enhance food value but can specifically target some health problems [37–42]. Scrutinizing the effect of probiotics on lipid metabolism and obesity is a burning issue, being currently discussed in the scientific literature [43–45]. Probiotic bacteria can improve the host’s health in many ways, including boosting the immune system by forming lymphocytes in many organs [46]. Lactobacillus paracasei is a species of lactic acid bacteria often used in fermented dairy and vegetable products and it is hosted in the human intestinal tract and mouth as a beneficial bacterium with health benefits [47]. It was reported to boost the immune system, probably enhancing cytokines’ production, making children less likely liable to common infectious diseases [48]. Moreover, it was confirmed to boost the activity of natural killer cells, lymphocyte proliferation, and antibody production. Consequently, it ameliorated the symptoms of influenza virus infection in mice and increased the survival rate of mice infected with E. coli, and also has anti-inflammatory properties [49]. Since this bacterium is isolated from a traditional fermented dairy product and based on its measured high lipolytic activity, it can be a good candidate to counteract metabolic syndrome while keeping the health status of the beings at its bioactive status. The postbiotic action on lipid metabolism may directly emit from the postbiotic’s lipolytic action, limiting the formation of complex lipid forms or indirectly through influencing the activity of peroxisome proliferator-activated receptor α (PPARα), which plays a crucial role in controlling lipid metabolism [50]. Besides the aforementioned benefits of Lactobacillus paracasei, and being isolated from a traditional fermented dairy product and based on its measured high lipolytic activity, it can be a good candidate to counteract metabolic syndrome while keeping the general health status. Probiotic cell-free extract (postbiotic) favors replacing the use of live cells to avoid bacterial contamination problems. Cell-free probiotic extracts can be safely used as a potent antioxidant ingredient in the nutritional and medicinal industries [51]. Hence, the current study investigates the lipolytic Lactobacillus paracasei postbiotic capacity to regulate lipid metabolism and target metabolic syndrome as a safer anti-obesity anti-dyslipidemia agent replacing ATOR and other drugs with reported or suspected health hazards.

2. Results 2.1. Information of Lactobacillus Paracasei and Its Extract Identifying the studied bacterial isolate by Matrix-assisted laser desorption/ionization ﬂight mass spectrometry (MALDI-TOF-MS) revealed the data in Table1. The obtained data refer to high probability of Lactobacillus paracasei ssp paracasei DSM 2649 DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), based on the rank quality and score value (2.058). The second ranked probability was assigned to Lactobacillus paracasei Molecules 2021, 26, 472 3 of 22

ssp paracasei DSM 20,006 DSM with a score value of 1.882. Less probabilities were found with other bacteria.

Table 1. Bacterial identiﬁcation using Bruker Daltonik matrix-assisted laser desorption/ionization (MALDI) Biotyper.

Rank (Quality) Matched Pattern Score Value NCBI Identifier Lactobacillus paracasei ssp paracasei 1 (++) 2.058 1288 DSM 2649 DSM Lactobacillus paracasei ssp paracasei 2 (+) 1.882 1288 DSM 20,006 DSM Staphylococcus xylosus DSM 3 (+) 1.743 1288 20266T DSM Staphylococcus saprophyticus ssp 4 (−) 1.597 147,452 saprophyticus DSM 20229T DSM Staphylococcus saprophyticus ssp 5 (−) 1.591 147,452 saprophyticus DSM 20,038 DSM 6 (−) Staphylococcus xylosus FI FLR 1.57 1288 Staphylococcus succinus ssp casei 7 (−) 1.55 201,828 DSM 15096T DSM Staphylococcus epidermidis 8 (−) 1.485 1288 10,547 CHB Staphylococcus xylosus DSM 9 (−) 1.457 47,714 20,267 DSM Staphylococcus xylosus DSM 10 (−) 1.456 47,714 6179 DSM Meaning of Score Values: Green color with the symbol (++) and the score range from 2 to 2.299 refer to secure genus identification, probable species identification. Yellow color with the symbol (+) and the score range from 1.7 to 1.999 refer to probable genus identification. Red color with the symbol (−) and the score range from 0 to 1.699 refer to not reliable identification.

Testing the bacterial isolate (identified as Lactobacillus paracasei) against a spectrum of the prevailing antibiotics revealed its high sensitivity against most of them (kanamycin, chloramphenicol, gentamycin, vancomycin, trimethoprim/sulphamethoxazole, cefotaxime, and streptomycin), while it had moderate sensitivity to erythromycin. Testing the hemolytic activity of Lactobacillus paracasei isolate did not exhibit any change in the zone surrounding the colony on the freshly prepared blood agar and it was defined as the safe γ-hemolysis. The data in Figure1 reflect general biochemical and biological information on the cell-free extract (postbiotic) of Lactobacillus paracasei. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoretic pattern of postbiotic showed two major protein bands, 30 and 46 kDa, referring to the two major hydrolytic enzymes (protease and lipase, respectively). The postbiotic contained a considerable lipolytic activity amounting to 2395 µg/g/min of the dried extract. The total crude protein in the postbiotic was 275 µg/g. The antioxidant enzymes, SOD, CAT, and GSH-px, in the postbiotic showed reasonable activities, recording 8250 ± 246, 1325 ± 33, and 545 ± 13 U g−1, respectively. Postbiotic exhibited high antioxidant activities, i.e., DPPH and ABTS scavenging activities amounting to 68.5% ± 3% and 57.36% ± 2% respectively. and 54.12% ± 1.4 % reducing power (1 mg mL−1). The postbiotic showed general antibacterial activity against all the tested pathogenic bacteria (8 strains). Molecules 2021, 26, 472 4 of 22 Molecules 2021, 26, x FOR PEER REVIEW 4 of 22

FigureFigure 1.1. BiochemicalBiochemical information information on on LactobacillusLactobacillus paracasei paracasei cell-freecell-free extract extract (postbiotic): (postbiotic): electro- elec- trophoreticphoretic pattern pattern (A (),A biochemical), biochemical profile proﬁle (B), ( Band), and antibacterial antibacterial activity activity (C). (ValuesC). Values in (B in) and (B,C ()C are) presentedare presented as mean as mean± standard ± standard deviation deviation (SD) (SD) (n = ( 3).n = 3).

2.2.2.2. BiologicalBiological ExperimentExperiment 2.2.1.2.2.1. TheThe ChangesChanges inin SerumSerum BloodBlood LipidLipid Profile Profile TheThe data data in in TableTable2 2reveal reveal the the changes changes in in blood blood lipid lipid profile profile in in the the rats rats receiving receiving HFD HFD −1 andand treatedtreated with with either either ATOR ATOR (10 (10 mg mg kg kg−1)) oror twotwo differentdifferent levelslevels ofof postbiotic postbiotic (100 (100 and and −1 200200 mgmg kg kg−1). It is evidentevident thatthat totaltotal serumserum lipidlipid waswas significantlysignificantly ((pp< < 0.05)0.05)increased increased in in thethe non-treated non-treated group group (positive (positive control) control) as as compared compared to to the the negative negative control control (non-receiving (non-receiv- −1 HFD),ing HFD), having having a 55% a 55% increase. increase. ATOR ATOR (10 (10 mg mg kg kg)−1 could) could reduce reduce the the total total lipid lipid level level by by aboutabout 25%25% ofof the positive control. control. In In comparison, comparison, the the two two levels levels of ofpostbiotic postbiotic (100 (100 and and 200 −1 200mg mgkg−1 kg) could) could further further reduce reduce it to itgreater to greater extents, extents, i.e., i.e.,29% 29%and and34%, 34%, respectively. respectively. The −1 Thetotal total lipid lipid level level of the of therat ratgroup group receiving receiving postbiotic postbiotic (200 (200 mg mg kg− kg1) was) was significantly significantly (p < p (0.05)< 0.05) lower lower than than the the positive positive control control and and the the ATOR ATOR group. group. At At the the same time, itit waswas notnot significantly different from the negative control. A similar trend of changes can be observed significantly different from the negative control. A similar trend of changes can be ob- with serum triglycerides, which was increased by about 100% in the positive control and served with serum triglycerides, which was increased by about 100% in the positive con- reduced by ATOR, postbiotic 100 mg kg−1, and postbiotic 200 mg kg−1, by about 35%, 32%, trol and reduced by ATOR, postbiotic 100 mg kg−1, and postbiotic 200 mg kg−1, by about and 45% compared to the positive control, respectively. The level of serum triglycerides in 35%, 32%, and 45% compared to the positive control, respectively. The level of serum tri- the high-dose postbiotic (200 mg kg−1) group was also significantly (p < 0.05) lower than glycerides in the high-dose postbiotic (200 mg kg−1) group was also significantly (p < 0.05) the ATOR group and slightly higher than the negative control. lower than the ATOR group and slightly higher than the negative control. Table 2. Effect of lipolytic Lactobacillus paracasei postbiotic at different concentrations (100 and 200 mg/kg body weight/day) Table 2. Effect of lipolytic Lactobacillus paracasei postbiotic at different concentrations (100 and 200 mg/kg body weight/day) and atorvastatin (ATOR) at 10 mg/kg body weight/day on the lipid profile of hyperlipidemic Albino rats. and atorvastatin (ATOR) at 10 mg/kg body weight/day on the lipid profile of hyperlipidemic Albino rats. Total Lipid Triglyceride Total Cho HDL-Cho LDL-Cho VLDL-Cho Group Total Lipid Triglyceride Total Cho HDL-Cho LDL-Cho VLDL-Cho Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) ± c ± e ± d ± a ± d ± e NCNC 462.0462.0 ±13.8 13.8 c 136.4136.4 ±2.4 2.4 e 152.3152.3 ±3.6 3.6 d 54.754.7 ±0.9 0.9 a 70.470.4 ±4.7 4.7 d 27.327.3 ±0.5 0.5 e PC 718.3 ± 15.9 a 273.1 ± 2.3 a 292.7 ± 5.7 a 32.2 ± 1.3 d 205.9 ± 7.7 a 54.6 ± 0.4 a PC 718.3 ± 15.9 a 273.1 ± 2.3 a 292.7 ± 5.7 a 32.2 ± 1.3 d 205.9 ± 7.7 a 54.6 ± 0.4 a ATOR 534.7 ± 14.7 b 176.1 ± 2.0 c 174.6 ± 4.1 c 45.4 ± 0.8 b 93.9 ± 3.9 c 35.2 ± 0.3 c b c c b c c CFE-1ATOR 508.0 534.7± ±13.6 14.7b,c 184.8176.1± ±1.8 2.0b 198.4174.6± ±5.2 4.1b 40.845.4± ±0.9 0.8c 126.993.9± ± 4.23.9b 36.935.2± ±0.4 0.3b CFE-2CFE-1 473.7 508.0± ± 12.913.6c b,c 149.6184.8± ±1.6 1.8d b 177.6198.4± ±3.2 5.2c b 46.740.8± ±0.8 0.9b c 101.1126.9± ±3.7 4.2c b 29.936.9± ±0.2 0.4d b c d c b c d TotalCFE-2 Cho: Total cholesterol. 473.7 ± 12.9 HDL-Cho: High-density149.6 ± 1.6 lipoprotein177.6 cholesterol. ± 3.2 LDL-Cho:46.7 Low-density ± 0.8 lipoprotein101.1 ± cholesterol.3.7 VLDL-Cho:29.9 ± 0.2 VeryTotal low-density Cho: Total lipoprotein cholesterol. cholesterol. HDL-Cho: NC: High-density Negative control. lipopro PC: High-fattein cholesterol. diet. ATOR: LDL-Cho: High-fat dietLow-density + atorvastatin lipoprotein 10 mg/kg choles- body weight/day.terol. VLDL-Cho: CFE-1: High-fat Very low-density diet + lipase crudelipoprotein extract 100cholesterol. mg/kg body NC: weight/day. Negative CFE-2:control. High-fat PC: High-fat diet + lipase diet. crude ATOR: extract High-fat 200 mg/kg diet ± body+ atorvastatin weight/day. 10 Values mg/kg are body presented weight/day. as mean CFE-1:SD (n High-fat= 8). Mean di valueset + lipase within crude the same extract column 100 with mg different/kg body superscripts weight/day. letters CFE-2: are significantly different (p < 0.05). A total of 40 animals were divided into five groups (8 animals each). Postbiotic is the cell-free extract (CFE). High-fat diet + lipase crude extract 200 mg/kg body weight/day. Values are presented as mean ± SD (n = 8). Mean values

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Serum total cholesterol was significantly (p < 0.05) and considerably elevated in the positive control, achieving a 92% increase over the negative control. Treating the rats with ATOR (10 mg kg−1) and two levels of postbiotic (100 and 200 mg kg−1) attenuated the magnitude of this big increase, lowering it by 40%, 32%, and 39% of the positive control value, respectively. The levels of blood LDL-cholesterol significantly (p < 0.05) increased in the positive control (192% relative increase) over the negative control while treating the rats with ATOR (10 mg kg−1), and postbiotic (100 and 200 mg kg−1) reduced it by about 54%, 38%, and 50% of the positive control, respectively. The changes in VLDL followed the same trend. Conversely, the positive control showed a considerable reduction in the level of HDL cholesterol (−41%) relative to the negative control. Simultaneously, the treatments, ATOR (10 mg kg−1) and postbiotic (100 and 200 mg kg−1), raised it by about 28%, 20%, and 30% of the positive control, respectively. None of the treatments (ATOR or postbiotic) were in the significant (p < 0.05) HDL level of the negative control—they were slightly lower.

2.2.2. Changes in the Antioxidant Enzymes Activities The data in Figure2 indicate significant ( p < 0.05) decreases in the activities of the antioxidant enzymes (glutathione peroxidase, glutathione-s-transferase, glutathione reductase, SOD) in the obese group compared to the normal group. Both ATOR and postbiotic could recover from these reductions, exhibiting significant (p < 0.05) increases and restoring the normal rats’ enzymatic activity. On the contrary, the level of malondialdehyde (MDA) was significantly (p < 0.05) increased in the highly obese group and brought down to the significant (p < 0.05) level of the negative control by both ATOR and postbiotic.

2.2.3. Changes in Body Weight and Body Weight Gain The data in Figure3 show the highest body weight increase in the group solely receiving HFD. On the other hand, the group receiving ATOR could significantly (p < 0.05) diminish the rate of body weight increase to reach a level even lower than the negative control. The two groups receiving the bacterial postbiotic could further resist the body weight increase, maintaining it lower than the negative control and the ATOR group. The net body weight gain in the postbiotic-treated (low level) group was significantly (p < 0.05) lower than the negative control but not different from the ATOR group. However, the high postbiotic level group was significantly (p < 0.05) lower than the negative control and the ATOR group.

2.2.4. Changes in Liver Functions Figure4 shows that the biochemical indicators of the liver functions were significantly (p < 0.05) enhanced by the HFD, compared to the negative control. Treating the rats with ATOR did not correct this increase, but rather pushed the rat physiology towards even more significant (p < 0.05) increases. It can be concluded that ATOR negatively affected liver function not only as compared to the negative control but also as compared to the positive group receiving HFD. On the contrary, the two postbiotic treatments had significant (p < 0.05) reductions in the liver function indicators’ activities compared to the positive group solely receiving HFD. The high level of postbiotic significantly (p < 0.05) brought the activities of AST and ALT within that of those negative control, while the low level was slightly higher. In conclusion, ATOR deteriorated the liver function while postbiotic ameliorated them. Similarly, ATOR significantly (p < 0.05) increased the total, direct, and indirect levels of bilirubin, compared with the two controls, while postbiotic reduced them to the negative control level. MoleculesMolecules 20212021, 26, 26, x, 472FOR PEER REVIEW 6 of 22 6 of 22

Glutathione peroxidase SOD 200 60 a ab b b b bc 150 b c 40 c 100 d

20 (U/ mg protein) mg (U/ 50

0 0 10 20 M D A Glutathione-s-transferase

(U/ g tissue) g (U/ 8 15

6 a a b b b 10 4 c 5

2 protein) mg (nmol/ b b b b

0 0 NC PC ATOR CFE-1 CFE-2 120 Glutathione reductase Group 100

80 a ab ab b 60

mol/ g tissue) g mol/ c

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0 NC PC ATOR CFE-1 CFE-2

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Figure 2.2. OxidativeOxidative stress parametersparameters in in liver liver of of male male albino albino rats, rats, i.e., i.e., glutathione glutathione peroxidase, peroxidase, glu- glutathione-s-transferase,tathione-s-transferase, glutathione glutathione reductase, reductase, superoxide superoxide dismutase dismutase (SOD), (SOD), and malondialdehyde and malondialdehyde (MDA) after after 9 9 weeks. weeks. Group Group 1 received 1 received a normal a normal diet (Negative diet (Negative control: control: NC). Groups NC). 2–5 Groups received 2–5 a received high-fata high-fat diet, diet, where where group group 2 did 2 notdid receivenot receive any further any fu treatmentrther treatment (Positive (Positive control: control: PC). Group PC). 3 Group 3 −1 was treatedtreated with with atorvastatin atorvastatin (10 (10 mg mg kg −kg1),), while while groups groups 4 and 4 and 5 were 5 were treated treated with withLactobacillus Lactobacillus −1 paracasei postbiotic postbiotic (CFE-1 (CFE-1 and and CFE-2; CFE-2; at 100 at 100 and and 200 mg200 kg mg−1 ,kg respectively)., respectively). Values Values are presented are presented as mean ± ± SD ((nn = 8). Mean valuesvalues within within the the same same histogram histogram with with different different superscripts superscripts letters letters are signiﬁcantlysignificantly different different (p (

2.2.3. Changes in Body Weight and Body Weight Gain The data in Figure 3 show the highest body weight increase in the group solely receiving HFD. On the other hand, the group receiving ATOR could significantly (p < 0.05) diminish the rate of body weight increase to reach a level even lower than the negative control. The two groups receiving the bacterial postbiotic could further resist the body weight increase, maintaining it lower than the negative control and the ATOR group. The net body weight gain in the postbiotic-treated (low level) group was significantly (p < 0.05) lower than the negative control but not different from the ATOR group. However, the high postbiotic level group was significantly (p < 0.05) lower than the negative control and the ATOR group.

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(A)

(B)

FigureFigure 3. 3.Body Body weight weight curve curve (A )(A and) and final final body body weight weight gain gain (B) through (B) through a 9-week a 9-week experimental experimental periodperiod of of male male Wistar Wistar Albino Albino rats. Grouprats. Group 1 received 1 receiv a normaled adiet normal (Negative diet (Negative control: NC). control: Groups NC). 2–5 Groups received2–5 received a high-fat a high-fat diet, where diet, group where 2 didgroup not 2 receive did not any receive further any treatment further (Positive treatment control: (Positive PC). control: −1 GroupPC). Group 3 was treated 3 was withtreated atorvastatin with atorvastatin (ATOR) (10 (ATOR) mg kg−1 (10), while mg groupskg ), while 4 and groups 5 were treated 4 and with5 were −1 Lactobacillustreated with paracasei Lactobacilluspostbiotic paracasei (CFE-1 postbiotic and CFE-2; (CFE-1 at 100 and and 200 CFE-2; mg kg at−1 ,100 respectively). and 200 mg Values kg are, respec- presentedtively). Values as mean are± SDpresented (n = 8). Meanas mean values ± SD within (n = the8). Mean same histogram values within with different the same superscripts histogram with lettersdifferent are significantlysuperscripts different letters are (p < significantly 0.05). Number different of animal (p < equal 0.05). 40 Number animals (fiveof animal groups equal of 8 40 ani- animalsmals (five each). groups Postbiotic of 8 isanim theals cell-free each). extract Postbiotic (CFE). is the cell-free extract (CFE).

2.2.4. Changes in Liver Functions Figure 4 shows that the biochemical indicators of the liver functions were significantly (p < 0.05) enhanced by the HFD, compared to the negative control.

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120 2.0 AST Total 100 a 1.5 a 80

60 1.0 b b c c 40 c c d d 0.5 20

0 0.0 200 2.0 ALT Direct a 150 1.5

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Enzyme Enzyme (U/L) a 50 c 0.5 b c c c c c 0 Bilirubin concentration0.0 (mg/dL) 350 2.0 ALP Indirect 300 a 1.5 250

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0 0.0 NC PC ATOR CFE-1 CFE-2 NC PC ATOR CFE-1 CFE-2 Group Group

FigureFigure 4. Aspartate 4. Aspartate aminotransferase aminotransferase (AST), (AST), alanine alanine aminotransferase aminotransferase (ALT), alkaline (ALT), phosphatase alkaline phosphatase (ALP),(ALP), and and bilirubin bilirubin (total, (total, direct direct and indirect) and indirect) in the serumin the ofserum male of Albino male rats Albino in 5 experimentalrats in 5 experimental groupsgroups during during the the 9-week 9-week study study period. period. Group Group 1 received 1 received a normal a normal diet (Negative diet (Negative control: control: NC). NC). GroupsGroups 2–5 2–5 received received a high-fat a high-fat diet, wherediet, where group group 2 did not 2 did receive not anyreceive further any treatment further treatment (Positive (Positive −1 control:control: PC). PC). Group Group 3 was 3 treatedwas treated with atorvastatin with atorvastatin (10 mg kg (10−1 ),mg while kg groups), while 4 andgroups 5 were 4 and treated 5 were −1 withtreatedLactobacillus with Lactobacillus paracasei postbiotic paracasei (CFE-1 postbiotic and CFE-2;(CFE-1 at and 100 CFE-2; and 200 at mg100 kgand−1 ,200 respectively). mg kg , respec- Valuestively). are presentedValues are as presented mean ± SD as (n mean= 8). Mean± SD ( valuesn = 8). withinMean thevalues same within histogram the same withdifferent histogram with superscriptsdifferent letterssuperscripts are signiﬁcantly letters are different significantly (p < 0.05). different Forty animals (p < 0.05). were Forty divided animals into ﬁve were groups divided into (8 animalsfive groups each). (8 Postbiotic animals iseach). the cell-free Postbiotic extract is the (CFE). cell-free extract (CFE).

2.2.5. ChangesTreating in Serumthe rats Protein with LevelsATOR did not correct this increase, but rather pushed the rat physiologyThe data in towards Figure5 provideeven more information significant on the (p serum < 0.05) protein increases. levels inIt can different be concluded rats that receivingATOR eithernegatively ATOR affected or postbiotic. liver function It was observed not only (Figure as compared5A) that totalto the protein negative was control but significantlyalso as compared (p < 0.05) to reduced the positive in the group positive receiv controling(obese) HFD. On compared the contrary, to the negativethe two postbiotic control. ATOR did not change the level of total protein from that of the positive control. treatments had significant (p < 0.05) reductions in the liver function indicators’ activities However, the two levels of postbiotic could raise the level of serum total protein to be in thecompared same significant to the (ppositive< 0.05) levelgroup of thesolely negative receiving control, HFD. which The is significantlyhigh level of (p postbiotic< 0.05) signif- highericantly than (pboth < 0.05) the brought positive controlthe activities and the of ATOR AST group.and ALT Although within the that positive of those group negative con- wastrol, not while significantly the low (p level< 0.05) was different slightly from higher. the negative In conclusion, control in ATOR the level deteriorated of serum the liver albumin,function ATOR while reduced postbiotic it significantly ameliorated (p < 0.05). them. The Similarly, groups receiving ATOR postbioticsignificantly did not(p < 0.05) in- experiencecreased the such total, reduction direct, and and either indirect remained levels in of the bilirubin, level of thecompared negative with control the (low two controls, levelwhile of postbiotic) postbiotic or reduced further significantlythem to the (negativep < 0.05) increasedcontrol level. (high level of postbiotic). The changes in serum albumin are confirmed by SDS-PAGE electropherogram (Figure5B, band2.2.5. A). Changes in Serum Protein Levels The data in Figure 5 provide information on the serum protein levels in different rats receiving either ATOR or postbiotic. It was observed (Figure 5A) that total protein was significantly (p < 0.05) reduced in the positive control (obese) compared to the negative control. ATOR did not change the level of total protein from that of the positive control. However, the two levels of postbiotic could raise the level of serum total protein to be in the same significant (p < 0.05) level of the negative control, which is significantly (p < 0.05) higher than both the positive control and the ATOR group. Although the positive group

Molecules 2021, 26, x FOR PEER REVIEW 9 of 22

was not significantly (p < 0.05) different from the negative control in the level of serum albumin, ATOR reduced it significantly (p < 0.05). The groups receiving postbiotic did not experience such reduction and either remained in the level of the negative control (low level of postbiotic) or further significantly (p < 0.05) increased (high level of postbiotic). The changes in serum albumin are confirmed by SDS-PAGE electropherogram (Figure 5B, band A). The positive control (obese rats) showed a significant (p < 0.05) reduction in the level of serum globulin as compared to the negative control. In contrast, ATOR could partially recover this reduction, but serum globulin was still significantly (p < 0.05) lower than the negative control. On the other hand, both postbiotic levels could more effectively encounter the reduction in serum globulin, bringing it to the significant (p < 0.05) level of the Molecules 2021, 26, 472 negative control. The changes in serum globulin are also confirmed by SDS-PAGE9 ofelec- 22 tropherogram (Figure 5B, G = globulin).

FigureFigure 5. 5.Total Total protein, protein, albumin, albumin, globulin, globulin, ( A(A)) and and SDS-PAGE SDS-PAGE (B (B) in) in the the serum serum of of male male Albino Albino rats rats afterafter 9 9 weeks. weeks. GroupGroup 11 receivedreceived aa normalnormal dietdiet andand served as a negative control control (NC). (NC). Groups Groups 2–5 2–5 received a high-fat diet, where group 2 did not receive any further treatment (Positive control: received a high-fat diet, where group 2 did not receive any further treatment (Positive control: PC). PC). Group 3 was treated with atorvastatin (ATOR) (10 mg kg−1), while groups 4 and 5 were Group 3 was treated with atorvastatin (ATOR) (10 mg kg−1), while groups 4 and 5 were treated with treated with Lactobacillus paracasei postbiotic (CFE-1 and CFE-2; at 100 and 200 mg kg−1, respec- −1 Lactobacillustively). Values paracasei are presentedpostbiotic as (CFE-1 mean and± SD CFE-2; (n = 8). at Mean 100 and values 200 mgwithin kg the, respectively). same histogram Values with are presenteddifferent assuperscripts mean ± SD letters (n = 8).are Mean significantly values within different the ( samep < 0.05). histogram Forty animals with different were divided superscripts into lettersfive groups are significantly (animals each). different Postbiotic (p < 0.05). is the Forty cell-free animals extract were (CFE). divided G = globulin, into five PsA groups = post-albu- (animals each).min, A Postbiotic = albumin, is the and cell-free PA = pre-albumin. extract (CFE). G = globulin, PsA = post-albumin, A = albumin, and PA = pre-albumin. 2.2.6. The Changes in Serum Creatinine, Uric Acid, and Urea Levels The positive control (obese rats) showed a significant (p < 0.05) reduction in the level The data in Figure 6 demonstrate significantly (p < 0.05) increased levels of serum of serum globulin as compared to the negative control. In contrast, ATOR could partially creatinine, serum uric acid, and serum urea in the obese group compared to the negative recover this reduction, but serum globulin was still significantly (p < 0.05) lower than the control, which were further significantly (p < 0.05) increased by ATOR. On the other hand, negative control. On the other hand, both postbiotic levels could more effectively encounter the reduction in serum globulin, bringing it to the significant (p < 0.05) level of the negative control. The changes in serum globulin are also confirmed by SDS-PAGE electropherogram (Figure5B, G = globulin).

2.2.6. The Changes in Serum Creatinine, Uric Acid, and Urea Levels The data in Figure6 demonstrate significantly ( p < 0.05) increased levels of serum creatinine, serum uric acid, and serum urea in the obese group compared to the negative control, which were further significantly (p < 0.05) increased by ATOR. On the other hand, the two groups receiving postbiotic could correct this increase and achieved significantly (p < 0.05) lower levels of the three indicators compared to both the positive control and the ATOR group, attaining the negative control level in case of creatinine and urea. In the case of serum uric acid, only the high level of postbiotic achieved the significant (p < 0.05) level of the negative control. Molecules 2021, 26, x FOR PEER REVIEW 10 of 22

the two groups receiving postbiotic could correct this increase and achieved significantly (p < 0.05) lower levels of the three indicators compared to both the positive control and Molecules 2021, 26, 472 the ATOR group, attaining the negative control level in case of creatinine and urea.10 In of 22the case of serum uric acid, only the high level of postbiotic achieved the significant (p < 0.05) level of the negative control.

10 Creatinine

4 a

2 b

c c c 0 10 Uric acid 8 a

4 b c d d 2 Concentration (mg/dL) Concentration

Urea 60 a b

40 c c c

0 NC PC ATOR CFE-1 CFE-2

Group Figure 6. Creatinine, uric acid, and urea in the serum of male Albino rats during the 9-week study Figure 6. Creatinine, uric acid, and urea in the serum of male Albino rats during the 9-week study period. Group 1 received a normal diet and served as a negative control (NC). Groups 2–5 re- period. Group 1 received a normal diet and served as a negative control (NC). Groups 2–5 received ceived a high-fat diet, where group 2 did not receive any further treatment (positive control: PC). aGroup high-fat 3 diet,was treated where groupwith atorvastatin 2 did not receive (10 mg any kg− further1), while treatment groups 4 and (positive 5 were control: treated PC). with Group Lacto- −1 3bacillus was treated paracasei with postbiotic atorvastatin (CFE-1 (10 mgand kg CFE-2;), while at 100 groups and 200 4 andmg kg 5 were−1, respectively). treated with ValuesLactobacillus are − paracaseipresentedpostbiotic as mean (CFE-1 ± SD (n and = 8). CFE-2; Mean at values 100 and within 200 mgthe kgsame1, histogram respectively). with Values different are super- presented asscripts mean letters± SD (aren = significantly 8). Mean values different within (p < the 0.05). same Forty histogram animals with were different divided superscriptsinto five groups letters (8 areanimals signiﬁcantly each). Postbiotic different ( pis

Molecules 2021, 26, 472 the positive control (obese group) revealed moderate hepato-cellular degenerative11 of 22 changes, mainly hydropic degeneration besides characteristic fatty infiltration as manifested by macro- and micro-steatosis in a variable number of hepatocytes (35–55% of cells). The portal triads revealed moderate lympho-plasmocytic inflammatory reaction and mild multifocalbiliary proliferation. hepatocellular The necrotic HFD rat areas, group replaced treated with by lymphocytes ATOR showed and some macrophages. sections with The portalmultifocal triads hepatocellular showed a moderate necrotic lympho-plasmocytic areas, replaced by lymphocytes inﬂammatory and macrophages. reaction and mildThe biliaryportal proliferation. triads showed The a numbermoderate of lympho-plasm hepatocytes affectedocytic inflammatory by fatty degeneration reaction and was mild few, rangingbiliary fromproliferation. 20% to The 25%, number while of other hepatocytes cells were affected normal. by fatty Other degeneration sections revealed was few, a moderateranging numberfrom 20% of to hepatocytes 25%, while other demonstrating cells were normal. macro-steatosis. Other sections Sections revealed of the a mod- HFD raterate group number treated of with hepatocytes postbiotic demonstrating (100 mg kg− macro-steatosis.1) exhibited normal Sections histo-morphological of the HFD rat featuresgroup oftreated hepatic with parenchyma postbiotic (100 and mg stroma, kg−1) exhibited normal hepatocytes,normal histo-morphological cord arrangement, features and vascularof hepatic structures. parenchyma Examining and stroma, the sections normal ofhe thepatocytes, group receivingcord arrangement, HFD and and treated vascular with thestructures. high level Examining of postbiotic the (200sections mg kgof −the1) revealedgroup receiving normal HFD hepatic and parenchyma treated with inthe almost high alllevel parts. of The postbiotic portal triads(200 mg and kg its−1) contentsrevealed includingnormal hepatic portal pare veins,nchyma the central in almost veins, all hepatic parts. sinusoids,The portal Von-Kupffer triads and its cells, contents the hepaticincluding cords, portal and veins, the the hepatic central stroma veins, appearedhepatic sinus- with normaloids, Von-Kupffer histo-morphologic cells, the structures. hepatic cords, and the hepatic stroma appeared with normal histo-morphologic structures.

H&E × 100 H&E × 400 Plate 1 (NC) Plate 1

Plate 2 (PC) (PC) Plate 2

) 1 − Plate 3 (ATOR: 10 mg kg 10 (ATOR: Plate 3

Figure 7. Cont.

Molecules 2021, 26, 472 12 of 22

Molecules 2021, 26, x FOR PEER REVIEW 12 of 22

) 1 − Plate 4 (CFE 100 mg kg (CFE 100 Plate 4

) 1 − Plate 5 (CFE 200 mg kg (CFE 200 Plate 5

Figure 7. Histopathological photomicrographs of liver tissue (H&E × 100 and 400; A and B) at the Figureend of 7. theHistopathological experiment. Plate photomicrographs 1: Negative control, of Pl liverate 2: tissue Positive (H&E control,× 100 Plate and 3, 400; ratsA receivingand B) at the endhigh-fat of the diet experiment. and treated Plate simultaneously 1: Negative with control, 10 mg Plate kg−12: atorvastatin Positive control, (ATOR), Plate Plates 3, 4 rats and receiving 5: rats high-fatreceiving diet high-fat and treated diet and simultaneously treated with Lactobacillus with 10 mg kgparacasei−1 atorvastatin postbiotic (ATOR),(CFE-1 and Plates CFE-2; 4 and at 100 5: rats receivingand 200 high-fatmg kg−1, dietrespectively). and treated Postbiotic with Lactobacillus is the cell-free paracasei extractpostbiotic (CFE). The (CFE-1 blue arrows and CFE-2; refer to at 100 andhydropic 200 mg degeneration. kg−1, respectively). Yellow arrows Postbiotic indicate is the macr cell-freeo- and extractmicro-steatosis (CFE). The in a bluevariable arrows number refer of to hepatocytes. The red arrows refer to moderate lympho-plasmocytic inflammatory reaction in the hydropic degeneration. Yellow arrows indicate macro- and micro-steatosis in a variable number of portal triads and the black arrows mark mild biliary proliferation. hepatocytes. The red arrows refer to moderate lympho-plasmocytic inflammatory reaction in the portal3. Discussion triads and the black arrows mark mild biliary proliferation. 3. DiscussionThe susceptibility of the bacterial isolate of Lactobacillus paracasei to different studied antibioticsThe susceptibility initially proved of the its bacterial extract’s isolate safety ofinLactobacillus food applications. paracasei Theto absence different of studiedantibi- antibioticsotic resistance initially and proved hemolytic its extract’s activity safety is a safety in food prerequisite applications. for The selecting absence the of potential antibiotic resistanceprobiotic and strain, hemolytic Lactobacillus activity paracasei. is a safety Hence, prerequisite the defined for γ selecting-hemolysis the on potential fresh blood probiotic agar is another evidence of the bacterial isolate’s safety. strain, Lactobacillus paracasei. Hence, the defined γ-hemolysis on fresh blood agar is another The high crude protein level (15%) in the postbiotic of Lactobacillus paracasei may refer evidence of the bacterial isolate’s safety. to this component’s possible role (e.g., antioxidant and antibacterial). The two bands’ lip- The high crude protein level (15%) in the postbiotic of Lactobacillus paracasei may refer olytic and proteolytic activities were confirmed using tributyrin agar base plates and skim to this component’s possible role (e.g., antioxidant and antibacterial). The two bands’ lipoly- milk agar media respectively (data not shown), and were similar to other works [52–54]. tic and proteolytic activities were confirmed using tributyrin agar base plates and skim milk The initially observed postbiotic high antioxidant potential, i.e., DPPH and ABTS scav- agar media respectively (data not shown), and were similar to other works [52–54]. The enging activity, was further proven by the high enzymatic activities of SOD, CAT, and initially observed postbiotic high antioxidant potential, i.e., DPPH and ABTS scavenging GSH-px. These results are in line with those demonstrating that cell-free extracts (CFE) of activity, was further proven by the high enzymatic activities of SOD, CAT, and GSH-px. Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Lactobacillus reuteri, and Sac- These results are in line with those demonstrating that cell-free extracts (CFE) of Lactobacil- charomyces boulardii have significant dose-dependent radical scavenging activities [55]. lus acidophilus, Lactobacillus casei, Lactococcus lactis, Lactobacillus reuteri, Saccharomyces The antioxidant activity of postbiotic may impact metabolic syndrome sinceand it can be qual- boulardiiified ashave high significant compared dose-dependentwith the result of radical Xing et scavenging al. [56], who activities obtained [55 10%]. The to antioxidant75% activ- activityities for of CFE postbiotic of 10 different may impact Lactobacillus metabolic spp. syndrome The noticed since broad-spectrum it can be qualified antibacterial as high comparedactivity of with the thepostbiotic result ofmay Xing counteract et al. [56 metabolic], who obtained syndrome 10% by to inhibiting 75% activities the abundant for CFE ofGram-negative 10 different Lactobacillus bacteria impairingspp. The gut noticed function broad-spectrum [57]. antibacterial activity of the postbioticThe may lipid counteract profile data metabolic showed that syndrome ATOR’s by optimal inhibiting dose the attenuated abundant the Gram-negative lipid increas- bacteriaing effect impairing induced gut by the function HFD, [bringing57]. it near to but still higher than the negative control. Comparatively,The lipid profile the optimal data showed dose of that postbiotic ATOR’s exerted optimal more dose reducing attenuated actions the on lipid the increas-serum ingtotal effect lipid induced and triglycerides, by the HFD, such bringing that the it nearfinal tolevel but was still not higher significantly than the ( negativep < 0.05) differ- control. Comparatively,ent from the negative the optimal control. dose Reducing of postbiotic effects exerted of ATOR more and reducing postbiotic actions were onalso the noticed serum total lipid and triglycerides, such that the final level was not significantly (p < 0.05) different

Molecules 2021, 26, 472 13 of 22

from the negative control. Reducing effects of ATOR and postbiotic were also noticed on the levels of serum cholesterol, serum LDL-cholesterol, and serum VLDL cholesterol, where the effect of postbiotic (200 mg kg−1) was not significantly (p < 0.05) different from ATOR (10 mg kg−1). The elevating effect of ATOR (10 mg kg−1) on HLD was in the same significant (p < 0.05) level of postbiotic (200 mg kg−1). Generally, the postbiotic action on the lipid profile was more effective than ATOR at the optimal dose of each; however, equal potencies were spotted on cholesterols and HDL changes. Having higher or equivalent potency surpassing ATOR in managing dyslipidemia may attribute a distinctive advantage to postbiotic as a preferable substitute for the synthetic drugs. ATOR is highly effective and has a large spectrum of action, lowering both LDL-C and triglyceride levels [12,58], and is usually prescribed for the treatment of dyslipidemia and the prevention of cardiovascular disease and hypercholesterolemia, through correcting serum lipid profile [59] and reducing cholesterol biosynthesis. However, the potential adverse effects of ATOR on the hepatic functions [60] may justify the use of this approach. The action of postbiotic of the potential probiotic Lactobacillus paracasei agrees with a study [61] using a probiotic dairy food, including Lactobacillus acidophilus La5 causing decreases in serum LDL-cholesterol, triacylglycerol, and increasing HDL-cholesterol in Wistar rats. The observed equal potency of ATOR and postbiotic to correct the significant (p < 0.05) reductions in the antioxidant enzymes in the obese group may prove the in vivo capacity of the postbiotic to restore the normal level. This conclusion is supported by the result that both ATOR and postbiotic could significantly (p < 0.05) and equally reverse the increasing trend of MDA observed in the obese groups, enabling the use of the second as a substitute of the first. Increased biomarkers of oxidative stress and decreased antioxidant defenses, particularly SOD, have been noticed in blood samples of patients with metabolic syndrome [62,63]. This inverse association was previously reported between serum antioxidant molecules (carotenoid and vitamin C) and metabolic syndrome [64]. The in vivo antioxidant capacity of postbiotic is supported by its in vitro antioxidant capacity evidenced in the extract (Figure1). This biological antioxidant capacity of the postbiotic may prevent and treat metabolic syndrome health complications, imparting protection on the human body [65]. The two groups receiving postbiotic could resist body weight increase induced by HFD ingestion better than ATOR at the optimal dose of each, maintaining the negative control level or even lower rat body weight. The absence of a significant difference between the two postbiotic levels may indicate the low concentration’s sufficiency to achieve the intended body weight reduction. Consequently, to counteract obesity, either level of postbiotic can be used. The reported link between obesity and a broad spectrum of CVD, morbidity, and mortality [66] may accentuate the importance of applying this postbiotic in food applications. ATOR can specifically modulate the complex pathways involved in metabolism, inflammation, atherogenesis, insulin sensitivity, and adipogenesis [9]. The mechanism of postbiotic may be exerted through different pathways, including antioxidant influence, lipolytic action, and antimicrobial action. This result agrees with a study using probiotic-fermented soymilk, including Bifidobacterium bifidum, Lactobacillus casei, and L. plantarum, showing significant reductions in the HFD-induced body weight gain and hyperlipidemia [67]. Since ATOR was unable to reverse the increasing effect of the HFD on liver biomarker (AST, ALT, and ALP) and rather significantly boosted them (p < 0.05), it is concluded to negatively affect liver function. The significantly (p < 0.05) postbiotic-decreased liver biomarkers’ levels compared to the positive group may refer to a remedial and maintaining role on liver functions. The conflicting actions of ATOR and postbiotic are also manifested in the significantly (p < 0.05) increased levels of the total, direct, and indirect levels of bilirubin in the ATOR group, accentuating the effect of the HFD against highly reduced levels in case of the postbiotic. This distinction may support the preference of the postbiotic over ATOR in treating the metabolic syndrome. Molecules 2021, 26, 472 14 of 22

The hepatic protective action of postbiotic as contrasted to the harmful or negative effect of ATOR was also manifested in the changes of serum protein levels. While ATOR could not reverse the reducing effect of the HFD diet on the total serum protein, the postbiotic could raise it to the significant (p < 0.05) level of the negative control. ATOR significantly (p < 0.05) reduced the level of serum albumin, while the postbiotic either maintained it in the level of the negative control or further significantly (p < 0.05) increased it. In contrast, ATOR could only partially recover the significant (p < 0.05) reductions in serum globulin triggered by the HFD, while postbiotic could more effectively recover it to the significant (p < 0.05) level of the negative control, confirming its safety and security. The hepatic protective effect of the postbiotic of Lactobacillus paracasei agrees with the results reported [67] and the data that Lactobacillus paracasei was reported to improve hepatic steatosis [50]. ATOR could not significantly (p < 0.05) counteract the increased levels of serum creatinine, serum uric acid, and serum urea incurred by HFD ingestion. It rather increased them further. On the other hand, postbiotic could correct these increases, bringing them down to the level of the negative control (at the high postbiotic dosage). This demonstrates that postbiotic can maintain the renal functions opposite to ATOR, which might negatively affect them. High level of serum uric acid is associated with metabolic syndrome [64] and generally, kidney damage indicators are associated with both cardiovascular diseases and lipid metabolism disorders [68]. A meta-analysis of a big population [69] showed an association between metabolic syndrome components, i.e., obesity and hypertriglyceridemia and kidney disease indicators, e.g., proteinuria and albuminuria risk. Hence, ameliorating the kidney status by postbiotic may reflect this agent’s healing action on metabolic syndrome, preventing kidney damage escalation. The liver histopathology of HFD-receiving rats indicated some amelioration by ATOR and the number of hepatocytes affected by fatty degeneration was slightly reduced by about 20–25% and other cells were apparently normal. Treating the same rats with the low level of postbiotic resulted in normal histo-morphological features of hepatic parenchyma and stroma with normal hepatocytes, cord arrangement, and vascular structures, while the high one produced mostly normal hepatic parenchyma. Therefore, postbiotic is more effective and safer than ATOR in treating metabolic syndrome. These histopathological changes are in harmony with the quantitative markers, including liver functions. So, the postbiotic is rather protecting the animal organ functions (liver and kidney). The action of Lactobacillus paracasei postbiotic against metabolic syndrome might have occurred through its antioxidant or proteolytic activities. The action of postbiotic against metabolic syndrome might have occurred through its antioxidant or lipolytic activities. The probiotic bacteria (Bacillus natto) was reported to significantly reduce the body, epididymal fat, serum triglyceride, and total cholesterol by inhibiting fatty acid synthesis and promoting fatty acid catabolism through expressing some signal [70]. Antioxidant activity was also associated with anti-obesity actions [51]. Lipolysis is a catabolism process of triacylglycerols stored in cellular lipid droplets [71]. The action mechanism of the postbiotic on lipid metabolism may directly emit from the lipolytic action of the postbiotic, limiting the formation of complex lipid forms. This may be supported by the previous reports that Lactobacillus metabolic substances could reduce the levels of triglycerides and LDL cholesterol while increasing the level of beneficial HDL cholesterol in an obese mouse model [62]. Alternatively, the postbiotic action may have an indirect mechanistic pathway. Lactobacillus paracasei was reported to have a high ligand activity for peroxisome proliferator-activated receptor α (PPARα), which plays a crucial role in controlling lipid metabolism, reducing adiposity, and improving hepatic steatosis [50]. Alternatively, this obesity-reducing action of the postbiotic can originate from its evidenced antioxidant activity. This relation was previously pinpointed in a previous study showing that the antioxidant activity of polyphenols from orange juice can protect against DNA damage and lipid peroxidation, modified multiple antioxidant Molecules 2021, 26, 472 15 of 22

enzymes, and decreased body weight [72]. The possible mechanisms of the antioxidant activity modulating the obesity management may possibly follow different mechanistic pathways, including suppression of fat absorption from the gut, suppression of anabolic pathway, stimulation of catabolic pathways in adipose tissue, inhibition of angiogenesis in adipose tissues, inhibition of differentiation of pre-adipocytes, and reduction of chronic inﬂammation associated with adiposity [73].

4. Materials and Methods 4.1. Materials Diagnostic kits (Bio Meriêuex Laboratory Reagents and Products, Cairo, Egypt), i.e., atorvastatin calcium (ATOR), were kindly provided as gifts by the Egyptian International Pharmaceutical Industries Co.

4.2. Bacterial Strain Lactobacillus paracasei was isolated from a sample of Egyptian mish cheese, identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF-MS; Bruker Daltonic GmbH, Bremen, Germany) according to Biswas and Rolain [74]. A pure culture of the bacterial isolate was assessed for hemolytic activity [75] on 5% sheep blood agar incubated at 37 ◦C for 24 h. Hemolytic activity of Lactobacillus paracasei was determined by streaking the pure bacterial culture on the freshly prepared blood agar before incubation at 37 ◦C for 48–72 h. The hemolytic type was defined according to the status of the clear zone observed. α-Hemolysis was defined when the colony was surrounded by a greenish zone. When the colony was surrounded by a clear white zone, it was defined as β-hemolysis. When there was no change in the medium surrounding the colony, it was defined as γ-hemolysis. Lipolytic activity of Lactobacillus paracasei was screened on tributyrin agar base plates by incubating at 37 ◦C for 48 h and measuring the clear zones resulting from lipolysis, according to Reference [76].

4.3. Lactobacillus Paracasei Cell-Free Extract (Postbiotic) Preparation In a model study, Lactobacillus paracasei was grown in De Man, Rogosa, and Sharpe (MRS) at 37 ◦C for 18 h before harvesting the bacterial cells by centrifugation (6000× g, 10 min, 4 ◦C; Centurion Scientific Ltd., K2015R, Church Farm, Stoughton, Chichester PO18 9JL, United Kingdom). The pellets were washed twice with deionized water before resuspending in the same water. The bacterial counts in the final suspension were adjusted to 108 CFU mL−1. The intracellular cell-free extract was prepared by the method of cells (108 CFU mL−1) with 1 mg mL−1 lysozyme at 37 ◦C for 30 min, followed by ultrasonic disruption. Sonication was performed for five min intervals in an ice bath. After removing the cell debris by centrifugation (8000× g, 10 min, 4 ◦C, Centurion Scientific Ltd., K2015R, Church Farm, Stoughton, Chichester PO18 9JL, UK), the supernatant was obtained as the intracellular cell-free extract (postbiotic) of Lactobacillus paracasei, lyophilized and kept at −20 ◦C until being used for chemical analysis and in the animal experiment. The lyophilized Lactobacillus paracasei postbiotic was dissolved in distilled water (1 mg mL−1) and used for its biochemical characterization.

4.4. Lactobacillus Paracasei Cell-Free Extract (Postbiotic) Characterization 4.4.1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) To identify the presence of lipolytic and proteolytic factors in the postbiotic, SDS- PAGE was carried out. Twenty µL of the extract was mixed with 20 µL of SDS-loading sample buffer (SDS 4%, β-mercaptoethanol 3%, glycerol 20%, Tris HCl 50 mM, pH 6.8, and bromophenol blue traces), heated at 96 ◦C for 3 min, and a 10 µL (per lane) aliquot was electrophoresed on SDS-PAGE, using an electrophoresis unit (Bio-Rad, Hercules, CA, USA) according to the method of Reference [77]. Molecules 2021, 26, 472 16 of 22

4.4.2. Antibacterial Activity Lactobacillus paracasei cell-free extract was screened against Gram-positive and Gram- negative bacteria for inhibitory activity using the well diffusion assay [16] using Mueller Hinton Agar (MHA) plates. Each strain was spread uniformly onto individual plates using sterile cotton swabs. Wells of 6 mm diameter were made on the MHA plates using gel puncture. Aliquots (30 µL) of postbiotic (500 µg mL−1) were transferred onto each well of all plates. Negative control (sterilized distilled water) was carried out. The antimicrobial activity was recorded as growth-free inhibition zones (diameter) around the well [78].

4.4.3. Lipase Activity The crude enzyme was prepared from the culture broth after the separation of cells and particles, i.e., the postbiotic. The lipolytic activity of the crude enzyme preparation was determined according to Reference [79] by incubating the enzyme extract with the substrate (p-nitrophenylpalmitate: p-NPP) for 15 min at 37 ◦C and the activity was quantiﬁed calorimetrically by measuring the extent of substrate cleavage at pH 8.0.

4.4.4. Antioxidants Activity The reducing activity was assayed in the postbiotic following Reference [80] by mixing 1.0 mL of postbiotic (1 mg mL−1) with phosphate buffer (2.5 mL) and potassium ferri- cyanide (2.5 mL). The mixture was incubated at 50 ◦C for 20 min then combined with 2.5 mL trichloroacetic acid and centrifuged at 1000× g for 10 min (Centurion Scientiﬁc Ltd., K2015R, UK). The upper layer of solution (2.5 mL) was mixed with 2.5 mL distilled water (2.5 mL) and 0.5 mL freshly prepared ferric chloride solution and the absorbance was measured at 700 nm. Ascorbic acid (1 mg mL−1) was used as standard. A blank was prepared without adding a standard or test compound. The reducing power is calculated according to the following equation:

blank % increase in reducing power = Abs test − Abs blank × 100 (1) Abs

The radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was conducted according to Ramadan [81] with few modifications. Briefly, 1 mL of 0.2 × 10−3 M DPPH solution in ethanol was mixed with 0.8 mL postbiotic. The mixture was kept in the darkness for 30 min before measuring the absorbance at 517 nm using a UV-Vis spectrophotometer (Jenway 6305 UV-Vis Spectrophotometers, Bibby Scientific, Beacon Rd, Stone ST15 0SA, UK). The blanks contained MRS broth and DPPH solution. The following equation calculated the radical-scavenging activity of postbiotic:

test % inhibition = Abs blank − Abs blank × 100 (2) Abs

The radical scavenging activity was also evaluated against ABTS (2,20-azino-bis (3- ethyl benzothiazoline-6-sulphonic acid) as described in Reference [82]. An aliquot of postbiotic (100 µL) was mixed with 3.0 mL ABTS+ solution and incubated for 15 min at room temperature before measuring the absorbance at 734 nm. Distilled water was instead of postbiotic in the blank solution. The inhibition % was calculated by measuring the reaction absorbance (Abs) of the control and sample at 734 nm and using the following equation:

% inhibition = Abs control − Abs sample/Abs control × 100 (3)

4.4.5. Total Protein and Antioxidant Enzymes The crude protein was estimated in postbiotic by bicinchoninic acid assay (BCA assay), using a standard kit, as described in Reference [83]. The activities of superoxide dismutase Molecules 2021, 26, 472 17 of 22

(SOD), catalase, and glutathione peroxidase (GSH-px) were measured using standard kits according to References [84–86].

4.5. Animal Experiment 4.5.1. Animals Healthy adult Wistar albino male rats, nearly the same age (2 months old), 160 to 170 g weight, were brought from the farm of the Faculty of Veterinary, Benha University (Egypt). Animals were kept under optimal conditions, i.e., 25 ◦C, 50% relative humidity, 12 h light/dark cycle. They were allowed free access to water and a standard diet containing (amount g/kg diet) corn starch 620, casein 140, sucrose 100, corn oil 45, mineral mix 35, vitamins mix 10, and Fiber 50 [87]. The biological experiment’s design and procedures got the approval of the Institutional Animal Care and Use Committee, Zagazig University (ZU-IACUC/3/F/175/2019).

4.5.2. Design After two-week acclimatization, a completely randomized design was followed to allocate forty animals into five groups of 8 animals each [88]. The five treatments were as follows: Group 1 (Negative control): Rats were fed on the basal diet (healthy control) without any treatments. Group 2 (Positive control): Rats were fed on the high-fat diet (HFD) containing 20% lard fat, 1% cholesterol, and 0.25% bile acid, integrated into the powdered standard animal diet. Group 3: (ATOR group) Rats were fed HFD and treated daily with oral administration of ATOR aqueous solution (10 mg kg−1) through gastric gavages for nine weeks as a standard antihyperlipidemic drug. Groups 4 and 5: (Treatment groups) Rats were fed HFD and treated daily with postbiotic (cell-free extract; CFE-1 and CFE-2; 100 and 200 mg kg−1 animal weight) through oral gastric gavages for 9 weeks, respectively. Initial and final body weights of all animals were measured at the start and after nine weeks of treatment.

4.5.3. Blood Sample Collection and Biochemical Parameter Assays At the end of the experiment, the rats were fasted for 12 h and blood samples were taken from the retro-orbital plexus veins by ﬁne capillary heparinized tubes, and allowed to clot before separating serum by centrifuging at 1008× g for 15 min. The serum was used immediately for the determination of all serum parameters. The activities of liver enzymes: alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), as well as serum total protein and serum albumin were determined according to References [89–91]. Serum globulin was calculated by subtracting the albumin from serum total protein. Total bilirubin was determined following Reference [92] and kidney function parameters: urea, uric acid, and creatinine, were also assayed according to Reference [93]. Lipid proﬁle, including total lipids (TL), triglycerides (TG), total cholesterol (TC), and high- density lipoprotein cholesterol (HDL-C), was determined following References [94–96]. Low-density lipoprotein cholesterol (LDL-C) was calculated as follows [97]:

LDL − C = TC − (HDL + TG/5) (4)

Very low-density lipoprotein cholesterol (VLDL-C) was calculated as TG/5.

4.5.4. Assessment of Hepatic Oxidative Stress Liver samples were washed immediately with ice-cold saline to remove excess blood and homogenized in cold 0.1 M potassium phosphate saline (pH = 7.4) at an extraction ratio of 1:9 w/v. The homogenate was centrifuged at 2800× g for 10 min at 4 ◦C (Cen- turion Scientiﬁc Ltd., K2015R, UK), and the supernatant was analyzed for antioxidant Molecules 2021, 26, 472 18 of 22

markers. Glutathione peroxidase (Gpx) was measured spectrophotometrically using Ell- man’s reagent [(DTNB) 5,50-dithiobis-(2-nitrobenzoic acid)] [85]. Glutathione-S-transferase activity was determined by monitoring change in absorbance due to thioether formation [98]. Reduced glutathione was assayed [99]. Lipid peroxides (malondialdehyde, MDA) were estimated following Reference [100] and superoxide dismutase in liver homogenate according to Reference [84].

4.5.5. SDS-PAGE of Serum Proteins SDS-PAGE (3% and 12%) of serum proteins was performed on a discontinuous buffered system [77], as explained before.

4.5.6. Histopathological Liver Examination The neutral formalin (10% v/v) was used to preserve liver specimens, as processed in an automated tissue processor [101]. Stained sections were examined for any pathological changes.

4.5.7. Statistical Analysis Data were subjected to analysis of variance (ANOVA) and one-way model statistical analyses using the software SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). All treatments and analyses were performed in triplicate and all mean values and standard deviations (SD) were calculated using Microsoft Excel software. Duncan’s new multiple-range test was used to resolve the difference between treatment means. Data were expressed as mean ± SD.

5. Conclusions Lactobacillus paracasei postbiotic with a high crude protein content (27.5%), a considerable lipolytic activity (2395 µg/g/min of the dried extract), high DPPH and ABTS scavenging activities, high antioxidant enzymes activities (SOD, CAT, and GSH-px), and broad- spectrum antibacterial activity may probably correct the disruption in lipid metabolism induced by HFD. It might also reverse the reductions in the antioxidant enzymes caused by ingesting HFD more effectively than atorvastatin, at the optimal dose of each. It might also counteract the body weight increase induced by the HFD more effectively than ATOR while avoiding the adverse effects on liver and kidney functions reported for ATOR. So, postbiotic may be used as an agent controlling and counteracting metabolic syndrome as efﬁciently as ATOR, while probably avoiding its secondary side effects, particularly on liver and kidney functions. The action mechanism of the postbiotic on lipid metabolism may be directly based on its lipolytic action, limiting the formation of complex lipid forms and fat accumulation or on its high antioxidant activity. These two mechanisms may protect against DNA damage and lipid peroxidation, leading to decreased body weight, probably through suppressing fat absorption and stimulating catabolic pathways in adipose tissues. Based on the lipolytic and antioxidant activities of Lactobacillus paracasei postbiotic, resulting in ameliorating lipid metabolism, it can be recommended as a natural effective agent treating or counteracting metabolic syndrome.

Author Contributions: Conceptualization, A.O., A.E.-H. and M.S.; Data curation, N.E.-G.; Formal analysis, T.N.A.; Investigation, A.O., N.E.-G., T.N.A., A.E.-H. and M.S.; Methodology, A.O., N.E.-G., T.N.A. and A.E.-H.; Project administration, M.S.; Resources, T.N.A. and A.E.-H.; Software, N.E.-G.; Writing—original draft, A.O. and A.E.-H.; Writing—review and editing, M.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Benha University, Zagazig University, and King Saud University. This research project was also supported by a grant from the “Research Center of the Female Scientiﬁc and Medical Colleges”, Deanship of Scientiﬁc Research, King Saud University, Riyadh, Saudi Arabia. Molecules 2021, 26, 472 19 of 22

Institutional Review Board Statement: The biological experiment’s design and procedures got the approval of the Institutional Animal Care and Use Committee, Zagazig University (ZU-IACUC/3/F/ 175/2019). Informed Consent Statement: Not applicable. Data Availability Statement: Data are available on request. Acknowledgments: The authors would like to thank Benha University and Zagazig University for financially supporting this project. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Not available.

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