CHARACTERIZATION AND PRODUCTION OF GOAT MILK KEFIR-PEPTIDE ON TRIGLYCERIDE SYNTHESIS OF CELL MODEL OF 3T3-L1

Goat milk kefir (GMK) refers to fermented products, generated through fermenting milk with microbial culture called kefir grains. Prior studies have reported the changes in kefir quality properties during aging process in which fermentation continues, resulting in changes of the peptide content. This research aims to investigate the effect of aging time on GMK–peptide characteristics to inhibite triglyceride syntheses on cells model of 3T3-L1. In this study, GMK were stored at 4 ± 1 o C for 0, 2, 4, 6 and 8 weeks, respectively. The protein profile was characterizedby implementing SDS-PAGE. The result of experiment indicated no protein degradation during the 6 weeks of aging period particularly for high molecular weight at 84 kDa, 80 kDa and 65 kDa. The GMK-supernatant from 8 weeks storage was performed by applying ultrafiltration membrane cut off < 30 kDa (UFC8 030.08). GMK-filtrate fractions (GMK-peptide) was collected as peptides. After 8 weeks of aging period, kefir protein profile was found to consist of peptide fractions and amino acid. The proteolytic activity of kefir grain increased linearly along with aging time (0-8 weeks). During aging period, the proteolytic activity of grain kefir released peptides and amino acid. In particular, the antioxidant activities were found significantly different (p<0.05) during aging periods. The antioxidant activities of GMK-peptide increased along with the elevating peptide concentration, from 3.30 % to 55.73%, with derivate by GMK-peptides of 2.75 – 10.39 mg/ml. Obesity associated with adipocyte hypertrophy occurred when TG accumulation.GMK-peptide of 100  g/ml indicated the lowest TG level (2.00 ± 0.03 mg/dl). This finding was in line with the inhibition of TG synthesis (61.14 ± 3.26 %). However, GMK-peptide contained antioxidant potency due to may be corelated with decreasing TG synthesis.The study thus suggested the important role of kefir to prolong aging in generating higher peptide bioactive as antioxidant and inhibition of TG synthesis of cell model of 3T3-L1.


INTRODUCTION
Goat milk showhigher digestibility then cow milk, distinct,higher buffering capacity (alkalinity) andtherapeuticeffect (Getaneh et al., 2016). Goat milk kefirrefers to milk culture, acidified by Lactic Acid Bacteria (LAB)such as Lactobacillusacidophilus, Lb. casei, Lb. reuteri, Lb. plantarum, Lb. bulgaricus, L. lactis, Bifidobacterium bifidum,B. lactis(Pacheco Da Silva et al., 2016). Free radicals is excess of oxidationreported to be one of thecauses ofpathogenesis and diseases in humans such asmembrane lipids,proteins, enzymes and DNAdamage (Rahal et al., 2014). It is consequentlyto prevent free radicals furtherreacted withliving cells (Chen et al., 2021). Antioxidants as scavenger to freeradical result of oxidation process.Despite exhibiting antioxidant activity of administration of artificial antioxidants against several oxidation systems, is restricted due to the potential risks for the living body. Variousantioxidantsources frequent consumptionlikevegetables,fruit,herbs andspices (Mushtaq, et al., 2020). Similarly,GMK-peptide as peptide short chain, and amino acidexhibited antioxidant properties (Zulleta, et al., 2009;Ceballos et al., 2009). Prior studies (Mahdi, Untari and Padaga, 2018) have identifiedantioxidative peptides from GMK as well asreported by Li et al., (2013). However, only few reports presented the bioactive peptides derived from GM-kefir exhibiting antioxidative activity during fermentation with grain kefir. GMK-Peptide particularly by applying ultrafiltration membrane cut off < 30 kDa (UFC8 030.08) fraction implemented in the research was isolated and characterized. Most of studies focused on HPLC fractionation for peptide characterization. This study additionally emphasizes that peptides were found novel due to its antioxidative activity relationship with TG synthesis in cells 3T3-L1 adiposity Obesityis identified asa chronic disease and defined as the accumulation of fat in the body, leading to various diseaseas hypertension, heart disease,osteoarthritis anddiabetes (Marseglia,et al. 2015). Proliferated cases of obesity emerges in various countries worldwide; thereby alleged as a global pandemic (Francisco et al., 2018), indicating a continuous escalation from year to year.The prevalence of obesity varies greatly among countries influenced by genetic and diet factors (Mohammadi, et al., 2019), inducing cascade reaction of oxidative stress and increasing reactive oxygen species (ROS), by accumulation of triglyceride (TG). Particularly, the prevalence of obesity in Indonesia adult men (> 18 years) was 7.8% (2018), and 19.7% (2013) (Riskesdas, 2018). Whereas in adult women (> 18 years), the prevalence of obesity increased from17.5% to 32. 9 (Panchal, et al., 2020).
Numerous studies have been conducted to prevent and treat obesity, through inhibiting adipocyte differentiation, stimulating exercises, reduce food intake, regulating lipid metabolism, and inhibiting lipase enzyme (Yun, 2010).
GM-Kefir and its active chemical constituents are reported to contain beneficial property and activity for the reducing of obesity. Therefore, applying kefir is take consideration effective as natural medicine with high safety compared to synthetic drug.Kefir from fermented goat milk contains high bioactive compound such as peptides (Fan, et al., 2018) research reports have indicated that peptides from GMK play in reducing body fat and lipid metabolismcontrol (Zeybek et. al., 2019). Purified tripeptide exhibited inhibition adipogenesis of 3T3-L1 with IC50 value of 14 g/ml. Similarly, flavoenzyme hydrolysate from soy protein isolate possessed a lipolysis-stimulating activity by measuring glycerol release in 3T3-L1 adipocytes. Further, GMK-peptide was reported to contain anti-hypertension and antioxidant. However, effort to update antiobesity peptide derived from GMK-peptide is not identified yet. Thus, this study aims to identify GMK-peptide as antioxidants and inhibitory effect of TG accumulation in cell model of 3T3-L1.

Whey Kefir Protein Profile Based on the Gel Electrophoresis
GM-Kefir was supplied by Bio-health small industry kefir production in Malang, Indonesia. The samples were stored at 4-6 o C for 0-8 weeks as aging period. After 8 weeks, GMK-kefir was centrifuged at 4500 rpm for 15 minutes to collect the supernatant of whey protein, performed by utilizing SDS-polyacrylamide gel electrophoresis to characterize the GMK-whey profile before and after aging period for 0-8 weeks.Gel polyacrylamide with eluent buffer consisted of 0.112 M acetic acid, 0.112 M Tris, and pH of 6.5, and buffer strip in 3% agarose with 0.20 M tricin, 0.20 M Tris, 0.55% SDS with pH of 8.1.Molecule standard content indicated that sample solution containing 15 l of each sample buffer and run in 4% Bis-Tris for 2 hours at 120 V of voltage and 4 o C of temperature. The relative band protein fraction migration was measured to determine the molecule weight (MW), by comparing with MW bandmarker line (Walker, 1996).

Peptide Determination
Upon the completion of a 8 week storage, GMK-peptide was fraction from GM-kefir by centrifugated at 4500 rpm for 15 minutes, then the supernatant was performed using ultrafiltration membrane cut off < 30 kDa (UFC8 030.08). Further, kefir filtrate fractions (GMK-peptide) was collected, dried freeze and stored at -20 ο C for later activity assay.

Determination of Amino Acid
In this study, amino acid were measured by HPLC (Knauer) according to Marino, et al (2010) with some modifications, including HPLC condition: Eurospher 100-5 C18 column of 250 x 4,6 mm with prep-column of P/N; 1115Y535; Eluent A: 0.01 M Acetic buffer with pH of 5.9 and B: MeOH: 0.01 M Acetic buffer pH 5.9 and tetrahydro folic (THF) by ratio (80:15:5); and Detector fluorescence (Excitation.  340 nm; Emission 450 nm). The sample elution was performed at a flow rate of 1.5 mL/min. In detail, 5 g of sample was prepared by weighing and inserting into a tube to be covered. Hydrochloric acid 6 N was added as much as 10 ml and vortex until exhibiting homogenous condition.
Hydrolysis of sample was performed by using autoclave at 110 o C for 8 h.Sample was cooled at room temperature and neutralized with NaOH 6 N.Solution of 40% Pb (CH3COO)4 and 15% oxalic acid were added and inserted into the vial and diluted with aquadest until 50 ml. Further, approximately 3 ml of sample was obtained and filtered with 0.45 m cover.Sample was later diluted for 10 times and incubated for 3 min in OPA. Sample was injected with as much as 30 l to HPLC (Nurliyani et al., 2014) Antioxidant activity of DPPH + free radical scavenging activity assay Antioxidant activity of DPPH + freeradical was determinate by Mc Cue and Shetty (2005) was considered to evaluate the antioxidant activity of the bioactive compounds, by measuring the decrease in absorbance of DPPH + at 517 nm (Daichi spectrophotometer UV-2000-1), which was in accordance with Mc Cue and Shetty (2005) with some modifications, included the addition of 4.5 ml methanol to 0.5 ml of DPPH + solution (about 0,5 g/L) and 0,1 mL of purified sampel.Standard index such as BHT was used as a reference index to determine radical scavenging capacity of the obtained sample, calculated as: Radical scavenging activity (%) = (At0 -Atend)/At0 X 100% Where : At0 refers to theabsorbance at time zero Atend refers to theabsorbance after 60 min.

Triglyceride (TG) Measurement
The 3T3-L1 adipocytes were harvested in 7 days after initiation of differentiation, treated with (GMK-peptide) consisting of positive (3T3-L1 with differentiation Cocktail) and negative control (3T3-L1 without differentiation Cocktail), the experiment consist of3T3-L1 with differentiation Cocktail and each sample was adding with GMK-peptide 25, 50, 75 and 100 g/ml sequentially. The cells were washed once with cold PBS.Cells were collected, and lysed in 100 l of lysis buffer (K610-100-2) cover to prevent evaporation. The cells were plate-heated at 90-100 o C for 30 min generating cloudy solution during heating process. Later, the process was progressed by plate cooling the solution to room temperature.The solution was further mixed by shaking plate for 1 minute. TG completely dissolved in the lipid extraction buffer, transferring 50l of lipid extracts to 96 wells plate added with 50 l of the reaction mix to each well containing TG standard, sample and control. Upon mixed, the solution was incubated at 37 o C for 30 minutes to measure the OD 570 nm (Kim, et al., 2006). TG concentration was calculated with: C=Ts/Sv (nmol/ul), where Tsdenotes TG amountfrom standard curve (nmol), Sv denotes the sample volume (before delusion) added in sample wells (l).

Statistical Analysis
In this study, data are presented as mean value with standard deviation.The significance between mean values was determined statistically with variance analysis in 5 different aging periods with 4 replications. Results of statistical analysis are reported as means ± standard deviation.

The Effect of Aging Period on Kefir Whey Protein Profile
According to the manufacturer of goat milk kefir manufacture, the final acidification degree of kefir should reach pH of 4.6. In this study,after 8 week aging period, kefir was centrifuged at 4500 rpm for 15 minutes.The supernatant is kefir whey containing soluble protein or peptide.Whey kefir protein indicated decreasing pH after 8 weekaging period affecting the protein fermentation andreleasing peptides. The pH degree of whey kefir protein could limit or modify the accessibility to some cleavage site for grain kefir enzymes (Kopf-Bolanz et al., 2014). The SDS-PAGE profiles of kefir whey are illustrated in Fig.1. This study however did not observe protein degradation during a 6 week aging period due to indicating similar protein profiles particularly for protein with MW at 84 kDa, 80 kDa and 65 kDa. Thus, it is suggested that this short fermentation did not exhibit protein degradation which could be seen by SDS-PAGE. However, after 8 week aging period, the protein profile indicated a decreasing high molecule weight band protein intensity. Through the fractionation process of whey protein by ultrafiltration, milk casein weredegraded into smaller fragments, despite leaving a remaining intact α-casein,β-casein andβ-lactoglobulin (Egger, et al., 2016). After 8 week aging period, the result indicated that kefir protein profile exhibited lower MW aspeptide fractions.  The decreasing concentration of high molecule weight component along with the increasing peptides in whey kefir protein indicated that kefir grain and their metabolites enzymes had fermented the protein further in aging at 4C.Kefir whey protein from different aging period contains various amounts of amino acid (illustrated in Table 1) such as, serine, isoleucine, phenylalanine, lysine and glutamic acid increasing duringaging period, as the result of composition variability generating various bioactive properties of GMKpeptides.

Effect of Aging on Proteolytic Activity
Milk protein are recognized as one ofsources for bioactive peptides. Table 1 show that several of amino acid increased significantly during aging periods, such as aspartic acid, glutamic acid, threonine, and arginine, but the other trend decreased. According to Panchal., et al (2020) and Wang et al., (2021) have been reported in goat milk and fermented goat milk products abundant of bioactive peptides. In additionkefir is known for its health promoting effects and there are more than 1500 peptides identified in milk kefir, reflecting the proteolytic activity of the microbial culture (Dallas et al., 2016). Proteolytic activity differs significantly (p<0.05) among aging period. The mean proteolytic activity of kefir culture increased linearly (r = 0,99) along with aging period (0-8 weeks) ( Table  2) depicted during aging period in releasing peptides and amino acid associated with proteolytic activity.

Antioxidant Activity of DPPH + Free Radical Scavenging Assay of GMKpeptide
Antioxidant activities of DPPH + free radical scavenging assay of GMK-peptide was presented in Table 2, indicating that antioxidant activities differed at (P<0.05) in aging periodfrom 0 to 8 weeks.The antioxidant activities of GMK-peptide increased simultaneously along with the increasing peptide concentration at 8 week aging period (from 3.30 % to 55.73% derivate byGMK-peptides of 10.39 mg/ml), which was also presented by Rahmawati and Suntornsu (2016), highlighting that antioxidative activity of 19%in goat milk yogurt increased during stirage at 4 o C up to 21 days.
However, this finding was in contrast with another study (Radiati, et al., 2012; indicating the decrease after 21 days of storage.Similarly, Panchal et al (2020) also measured ABTS activity of GMbevarageby microbial culture and additionwith grape pomace on gut microbiota and antioxidant activity. GMyogurtwith grape was 418.02 ± 16.14 mmol TE/g and goat milk with grape juice was 743.78. Chen et al (2019) investigated that cheddar cheeseshow the increasing antioxidants activity during ripeningin the first 4 weeks of storage (P <0.050, and reached the maximum of 43.86% to 47.43% which washigher thancontrol cheese. Such finding was in accordance with another study following the antioxidant activity of amino acids, released during the fermentation process such as cysteine, tryptophan and methionine (Elias et al., 2005).
This study, particularly, reported a significant increase of DPPH + free radical scavenging activity than those in control (0 week). Another study (Liu. et al., 2016) indicated GMK-peptide could extend the proteolysis with maximum antioxidant activity of peptides in this experiment to be 55.73±1.85 % ( Table 2).

Effect of GMK-peptide on Triglyceride Level on Cell Model of 3T3-L1
Cell model of 3T3-L1 refers to cell model of obesity. Obesity can induce oxidative stress leading to free radicals, thereby requiring antioxidants to reduce free radical levels. Fermented milk provides a source of antioxidants in the forms of protein derivatives with low molecular weight having antioxidant activity. In this study, fermented peptides were isolated by centrifugation and filtration to obtain GMKpeptides with molecular weights below 30 kDa, illustrated in Table 2. The finding presents that higher peptide concentration resulted in higher free radical scavenging in line with the inhibition of TG synthesis. GMK-peptide has antiinflammatory agent (Raras, et al., 2015), antioxidant agent, antiadipogenesis which are advantageous in preventing obesity.GMK-peptide (Table 3), inhibited of the transcriptional regulation of lipid synthesis or stimulation of lipolysis in 3T3-L1 adipocytes. The pre-adipocyte differentiated into adipocytes is regulated by a complex network of transcription factors. After differentiation, C/EBPβ was induced immediately, while C/EBPα and PPARγ serve as master regulators of adipogenesis which is critical to the progression of the final stages of adipocyte differentiation. In general, differentiation of adipocyte and fat accumulation are associated with theobesity development.
The increasing number of fat cells and adipose tissue mass could further cause obesity. Theoretically, the TG formation was measuredby optical density changing, thereby generating of lipid droplets formationwas measured by colorymetric assay, the higher TGresulting in higher absorbance, effective in the induction of differentiation of pre-adipocyte to adipocyte.
with negative control indicated "undifferentiated" as marked by the lowest TG level in colorimetric reading. There was difference between undifferentiated cells (Negative Control with the differentiated cells (Positive Control). In this research, GMK-peptide was evident to reduce TG level of 3T3-L1 (66.00 ± 2.06 %). Furthermore, higher TG level in adipose tissues increased the oxidation in cells level. Higher TG in plasma serum is correlated with obesity. In the current study, GMKpeptide was imminent to reduce TG level in cells of 3T3-L1 adiposity.Increasing theTG inducedthe fatty Acid Synthase (FAS),Acetyl-CoA Carboxylase (ACC) and adipocyte-specific fatty acid binding protein (aP2) genes expression. The decreasing TG content was resulted from the decreasing re-esterification of FFA. Peptide was also found to decrease PEPCK level, playing a role in adipogenesis through induced the ligand Peroxisome Proliferator-Activated Receptor γ (PPARγ) which contributed to induce of adipocyte-specific gene expression and differentiation by controlling the fat accumulation in the adipose tissue mass associated with obesity development. These results suggest that GMK-peptide contains anti-adipogenesis effect, lowering body weightas well as lowering FAS concentration in adipose tissue and serum of experimental rats. Thus, anti-obesity potency of GMK-peptide is strongly potential to its amino acid content based on HPLC assay. This finding is in accordance with another study (Kim et al., 2017) emphasizing that peptide exhibited antiobesity activity through antioxidant activity, also supported by other studies reporting the role of peptide in reducing lipid accumulation.

CONCLUSION
In sum, this study presented the role of kefir in addressing the obesity by investigating several components of kefir storage. It was also reported that aging period contributed to proteolytic activity following the increasing peptide and antioxidant activities to offer synergistic efficacy in blocking cellular oxidation mechanism. Subsequently, GMK-peptide was found to be potential in preventing fat accumulation through inhibiting molecular pathway of lipid.
M.Kes from Faculty of Medicine of Universitas Pajajaran, Bandung in providing adipocyte cells to support the research.