Stability Study of Fermented Milk using Lactiplantibacillus plantarum subsp. plantarum DAD-13 with Alpha Glucosidase Enzyme Inhibitory Activity on Simulated Gastrointestinal Digestion and Storage at Cold Temperature

. Fermented milk has bioactive peptides with alpha-glucosidase inhibitor capabilities. To be used widely, it must have stability when it stored and when it enters the human digestive tract. The purpose of this research was to understand the stability of alphaglucosidase inhibitory (AGI) activity of bioactive peptide from fermented milk on simulated gastrointestinal digestion and the stability during storage on cold temperature. Fermented milk was prepared by inoculating L. plantarum subsp. plantarum DAD-13 on sterilized milk for 18 h. The fermented milk was placed in simulated gastrointestinal digestion used consecutive pepsin for 3 h, bile salt and tripsin for 2h and tripsin for 8h. Determination of storage stability was carried out at cold temperature (4oC) for 30 days. Bioactive peptides simulated in the human digestive tract still had AGI abilities even though the values had decreased. The AGI activity of bioactive peptide of fermented milk decreased during digestion. AGI value decreased during storage but still had high inhibitory ability after 30 days (19,0126%). Thus, fermented milk could serve as an antihyperglycemic peptide source and alternative food for maintaining/reducing blood glucose.


Introduction
Diabetes is a chronic metabolic disorder that causes elevated blood sugar levels as a result of either inadequate pancreatic insulin synthesis or inappropriate body insulin metabolism.According to WHO [1] the age of death standard associated with diabetes has increased by 3% from 2000 to 2019, even the death rate from diabetes in development countries has increased by 13%.Stimulation of insulin secretion, increased action of insulin on target tissues, using oral hypoglycemic medications such as biguanides and sulfonylureas, use of meglitinides, thiazolidinediones, Glutation like Peptide-1 receptor agonists, SGLT2 inhibitors, exogenous insulin, DPP 4 inhibitors and inhibition of polysaccharides degradation by α-glucosidase enzymes are several types of treatment that have been applied to type 2 diabetes patients [2].Therapy using alpha glucosidase inhibitors (AGI) is one of the most widely used.The problem is, continuous use of drugs has side effects such as flatulence, diarrhea, dizziness, vomiting, other GI tract problems and liver disorders [3].Therefore, there is a need to search for alternative inhibitors of α glucosidase.
According to Stefano et al [4], peptides, non-starch polysaccharides, triterpenoids, and phenolic substances have a role in α-glucosidase inhibitory activity.Food ingredients with high peptide content are easy to find.Bioactive peptides are a component of proteins with biological functions that are advantageous to human health, such as: antimicrobial, antioxidant, antihypertensive, anti inflammatory and antidiabetic activities [4].According to Ibrahim [5], peptides with AGI capability are those that have 3-6 amino acid residues, consisting of lysine, threonine, serine, arginine or tyrosine at the N-terminus, proline residues close to the C-terminus; and at the C-terminal there is alanine or methionine.Meanwhile, according to Ren et al [6] hydrophobic amino acids, especially proline and leucine, contribute greatly to the activity of α-glucosidase inhibitors.
Milk contains amino acids proline and leucine, as well as high hydrophobic amino acids, so potential to produce peptides with AGI capabilities [7].Lactic acid bacteria (LAB)-fermented milk products have the potential to have a high AGI capability due to protein hydrolysis activity during fermentation.In this study, Milk is fermented using Lactiplantibacillus plantarum subsp.plantarum Dad-13 (L.plantarum Dad 13) an indigenous lactic acid bacterium found in dadih, a traditional Indonesian (West Sumatra) beverage made from fermented buffalo milk [26].This bacteria is known to have the ability as a probiotic, have proteolytic activity and other functional properties.
Meanwhile, according to Vander [8], protein digestion by digestive enzymes starts from the stomach to the small intestine.Protein digestion in the human digestive tract can affect the performance of bioactive peptides.According to Lin et al. [9], α-glucosidase produced in the small intestine mucosa plays a role in starch metabolism with different rates and amounts.Two types of α-glucosidases in the human brush border membrane include sucrase isomaltase (SI) and maltase glucoamylase (MGAM).Maltase glucoamylase is 2% of all proteins that are in the brush border, has maximum activity at 5-9 glucose residues, contributes to the activity of 20% maltase, a little isomaltase and all glucoamylase (substrate: limit dextrin and starch).While sucrase isomaltase is 10% of all proteins in the brush border.SI is evenly distributed in the small intestine, 20-30% smaller in the distal ileum and less abundant in the colon [10].To be able to act as AGI, bioactive peptides must be able to reach parts of the digestive tract where carbohydrate metabolism by the enzyme alpha glucosidase occurs, so that peptides from fermented milk with AGI activity must also be studied for resistance in the human digestive tract.In addition, based on Konrad [11] during fermentation and chilled storage, many of bioactive compounds such as peptides are released.This is possible as a result of the action of proteinases and peptides from lactic acid bacteria's proteolytic mechanism during cold storage, which releases and accumulates peptides with various molecular weights.No studies has yet discussed the stability of AGI bioactive peptides derived from milk fermented with L. plantaraum DAD 13 either in the digestive tract or in storage.Therefore, it is important to analyze the stability of AGI activity during storage.In this research, we analyzed the ability of AGI in fermented milk and the resistance to in vitro simulation of human digestion as well the stability during low-temperature storage.

Material
The LAB strain Lactiplantibacillus plantarum subsp.plantarum DAD-13 utilized in this work was from PSPG Gadjah Mada University culture collection and was grown in routine De Man Rogosa Sharpe (MRS) broth culture media (Merck).Skim milk (Lactona) was purchased from local market.α-glucosidase (AG) enzyme from Saccharomyces cerevisiae (freeze dried powder, type I, ≥10 units/mg protein) was obtained from Sigma Aldrich (Merck), o-phthaldialdehyde (OPA) was purchased from Merck.Analytical-grade chemicals were used for all other substances in this study.

Culture preparation
Prior to research, a preparatory stage was carried out.The preparation stage was aimed at ensuring the availability and homogeneity of lactic acid bacteria isolates during the study.The main material used for stock culture is L. plantarum DAD 13.According to Chen et al [30], the preparation step was completed by cultivating pure cultures of LAB in MRS (de Man, Rogosa, and Sharpe-Merck) broth medium at 37oC for 18 hours.The strain was cultured twice before being used for research.

Fermented milk preparation
The lactic acid bacteria starter was prepared by inoculating the bacterial culture into 10% skim milk (m/v) and incubating at 37 for 24 hours.Then it was recultured into 10% (m/v) skim milk to obtain a starter culture.The culture starter is then used for the production of fermented milk by culturing the starter into 12% (m/v) skim milk and incubating 18 hours at 37oC.

In vitro simulated gastrointestinal digestion
The procedure was conducted using method described by Muganga et al. [12] and Puspitojati et al [13] with modification.10 ml fermented milk sample were adjusted to pH 2,5 using 1.0 mol/l HCl and hydrolyzed with porcin pepsin (1:10.000)3g/l for 3 h at 37°C and shaking at 1.33 Hz in a water bath shaker (simulated gastric juice/SGJ).Then, pH was adjusted to 5 using 1 mol/l NaOH and added with Bile salt 3g/l and trypsin (1:250) 1g/l then incubated at 37°C for 2h (simulated duodenal juice/SDJ).Subsequently adjusted to pH 8 by adding 1 mol/l NaOH followed by the addition of trypsin (1:250) 1g/l and incubated at 37°C for 8h (simulated intestinal juice/SIJ).The reaction was stopped by dipping the sample into boiling water for 10 min, then cooled in an ice bath, and was used for further analysis.

Stability at cold temperature
Fermented milk was stored at cold temperature (4oC) in refrigerator for 30 days and analyzed every 6 days according to Paludetti et al [14].

Protein extraction
Each sample of fermented milk was centrifuged twice at 4000 g for 15 minutes to take the supernatant as a protein extract [15].

Survival rate
Survival rate is determined based on the method of Muganga et al [12].First, 1 mL of fermented milk (sample) was serially diluted in sterile saline (NaCl) with a concentration of 0.85%.Then, plated on MRS agar and incubated for 48 hours at 37°C in an anaerobic chamber to determine the number of viable cells.Survival rate is determined based on the following equation:

Determination of α-glucosidase inhibition (AGI)
Enzyme activity inhibition was carried out according to Zeng et al. [16] with minor modifications.This method is based on the reaction of the substrate with the enzyme which will produce a colored product.Preparation of acarbose reagents, enzymes, and p-Nitrophenyl-α-D-gluco pyranoside using 0.01M phosphate buffer saline (pH of 6.8).The reaction mixture consisted of 25 µL 10mM p-Nitrophenyl-α-D-gluco pyranoside, 25 µL 0.01M phosphate buffer saline pH 6.8, 25 µL acarbose 1% or sample.After that, it was incubated for 10 minutes at 37°C.Added 50 µL of α-glucosidase 1 U/mL, then incubated for 30 minutes at 37°C.A 100 µL addition of 0.1 M sodium carbonate solution stopped the reaction.Using a spectrophotometer with 405 nm wavelength, absorbance was measured.The blank solution was prepared without sample and the addition of enzymes, the positive control without sample but using the α-glucosidase enzyme, while the negative control only used 225 µL of phosphate buffer saline.The AGI activity was determined by the formula: (2)

Degree of hydrolysis
Degree of hydrolysis is determined based on Ramchandran and Shah [17].Based on the amount of free amino acids in fermented milk, the degree of hydrolysis was determined.After 5 minutes of vortexing with 3 mL of o-phthaldialdehyde in the filtrate (150 L), spectrophotometer was used to measure absorbance at 340 nm.The degree of hydrolysis was determined by comparing the readings on control milk (fermentation time 0) with that after fermentation.

Data analysis and statistics
Data measurement results are presented in the mean ± standard deviation (sd).The effect of treatment on variables was analyzed using analysis of variance (ANOVA).Duncan's test of multiple comparisons (IBM SPSS Statistics 27) was carried out to determine the difference in the average treatment between variables.Based on Table 1, the most significant reduction in survival rate occurred in simulated gastric juice (SGJ) from 1 to 3 hours.While in simulated duodenal juice (SDJ) and simulated intestinal juice (SIJ) there was a decrease and increase in the survival rate.The decrease in LAB viability in SGJ was caused by the low pH and the pepsin enzyme's presence..While the decrease in survival rate in SD is caused by the presence of bile salts and continuous stress on cells by the presence of SGJ which is continued by SDJ [18].Bile salts can have a damaging effect on the lipid membrane of bacterial cells which will affect the integrity of the cell wall and reduce the survival rate [19].The increase in survival rate in SIJ was probably caused by an increase in pH to 8. Thus, lactic acid bacteria L. plantarum DAD 13 in fermented milk have tolerance in GIT conditions.

Stability in simulated gastrointestinal tract conditions
Total peptide and degree of hydrolysis of fermented milk increased during SGJ, SDJ and SIJ.The increase occurred both in the decrease and increase in the survival rate of lactic acid bacteria because protein hydrolysis was carried out by both LAB and digestive enzymes.Gatric juice is in the stomach which contains pepsinogen (inactive form of pepsin) and hydrochloric acid (HCl) [8].In the stomach, food mixes with HCl and the enzyme pepsin.In this section, protein digestion first begins [8].Pepsin is the only proteolytic enzyme in the human stomach.
While the AGI value decrease during digestion simulation, it can be caused by excess hydrolysis of peptides in fermented milk by LAB and digestive enzyme.The size of peptide may not be suitable for inhibiting alpha-glucosidase activity.The size of these peptides is in accordance with AGI activity [5; 11;20].Peptides with 3-6 amino acids [5; 7; 21; 20] or peptides with a molecular mass of 3-10 kDa [11] are known to have important role as AGI.
Furthermore, hydrolysis through simulated GI track causes the formation of smaller sizes which are not suitable for AGI activity.Although AGI activity has decreased, fermented milk by L plantarum DAD 13 still has AGI activity after going through SGJ, SDJ and SIJ.There are differences in the stability of bioactive peptides, according to Liu and Pischetsrieder [23].Many bioactive peptides were released during the simulation in the digestive tract, especially following the simulation in oral, stomach, and intestine digestion, the concentration of the angiotensin-converting enzyme inhibitor β-casein grew up to 10,000 times.Soy protein-derived peptides with antihyperglycemic properties are partially absorbed across Caco-2 monolayers and boundary peptidases also hydrolyze them into shorter fragments [24].Peptides that are resistant to gastrointestinal digestion in vitro are related to their structure.In comparison to the unstable peptides, they are smaller in size and less hydrophobic, branched-chain aliphatic N-terminal residues, have a higher positive net charge at gut pH., no leucine at the C-terminus, and more proline and histidine content particularly at the C-terminus [25].
Several studies examined lactic acid bacteria (BAL) activity with AGI, including research conducted by Ramchandran and Shah [17] on L. delbrueckii ssp.bulgaricus 1092, L. acidophilus 33200, L. casei 2607 and B. longum 5022; Bajpai et al [27] on L. sakei 1I1; Panwar et al [28] on L.plantarum; Ramchandran and Shah [29] on S.thermophillus Eps producing strain; Chen, et al [30] on L. rhamnosus Z7, L. casei 2W; Zeng et al [16] on Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactobacillus rhamnosus; Muganga [12] on L. acidophilus CCFM6, L. plantarum CCFM47; and Sasikumar et al [31] on Lb. plantarum BR2.From the studies, it is not clear what components of BAL play a role in AGI and the influence of various growth factors on AGI.Some discuss related to bioactive peptides produced due to hydrolysis by the activity of proteolytic bacteria, some discuss the extracellular components of bacteria that play a role in AGI.So that AGI activity could have occurred due to a combination of bioactive peptides and extracellular components of BAL, but to prove this there must be further research.Survival rate of lactic acid bacteria and the pH value of fermented milk at low temperature storage (4 o C) are shown in Tabel 2. The pattern of the number of lactic acid bacteria and pH decreased with increasing storage time.This can happen because the activity of lactic acid bacteria produces higher lactic acid with longer storage time.The temperature spectrum of LAB's development is highly varied.They typically grow best at temperatures between 20 and 40 °C, generally mesophiles, with maximum temperature of around 50 °C.[22].Even though at low temperatures lactic acid bacteria are not in the best conditions for growth, they can still carry out their metabolism slowly so that the number of lactic acid bacteria and acidity still increase.The total peptide and the degree of hydrolysis increased with increasing storage time, while the AGI value decreased with increasing storage time.An increase in the degree of hydrolysis can occur because lactic acid bacteria can still carry out proteolytic activity even at low temperatures.As a result, the measured total peptide is higher.While AGI activity decreases, it can be caused by hydrolyzed peptides into smaller molecular weight peptides that are less effective as alpha-glucosidase inhibitors.According to Ren et al [6], α-glucosidase inhibitor activity belongs to peptides with 3-6 residues.The AGI activity value of all fermented milk was still below the acarbose (5 mg/ml) inhibitor activity value (50.56%).

Conclusion
AGI activity of fermented milk by L plantarum DAD 13 decreased after simulating the human digestive tract and storage at cold temperatures due to excessive hydrolysis by lactic acid bacteria and digestive enzymes.Milk fermentation using indigenous lactic acid bacteria L. plantarum Dad-13 can serve as an antihyperglycemic peptide source and alternative food for maintaining/reducing blood glucose because has AGI activity that is resistant to simulation of the human digestive tract and can withstand cold storage for 30 hours.Further research under different storage conditions and longer time is needed to better know the AGI stability of fermented milk.

Fig. 1 .
Fig. 1.Total peptide, degree of hydrolysis and AGI activity of fermented milk using L. plantarum DAD 13 at various storage time in refrigerator temperature (4oC)

Table 1 .
LAB survival rate, total peptide, degree of hydrolysis and AGI activity of fermented milk under simulated gastrointestinal tract conditions.

Table 2 .
LAB survival rate and pH of fermented milk during storage at cold temperature