Lactobacillus bulgaricus improves antioxidant capacity of black garlic in the prevention of gestational diabetes mellitus: a randomized control trial

Objectives: Lactobacillus bulgaricus may improve antioxidant capacity of black garlic in the prevention of gestational diabetes mellitus (GDM). Methods: Black garlic was prepared with or without L. bulgaricus. Volatile and polysaccharides were analyzed by using LC-MS, Fourier Transform infrared (FTIR) and 13C nuclear magnetic resonance (NMR). The study design was parallel randomized controlled trial and 226 GDM patients were randomly assigned into BG (black garlic and L. bulgaricus) and CG (black garlic) groups, and allocation ratio was 1:1. The treatment duration was 40 weeks. Fasting blood glucose (FBG) and 1- and 2-h blood glucose (1hBG and 2hBG) after oral glucose tolerance test (OGTT) were detected. Antioxidant function of black garlic was determined by measuring plasma malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) in GDM patients. The comparison between two groups was made using two independent samples t test. Results: The intake of nutrients was similar between two groups (P>0.05). L. bulgaricus promoted the transformation of the glucopyranoside to glucofuranoside. L. bulgaricus increased the abilities of black garlic for scavenging hydroxyl radicals, 2,2′-azino-bis (3-ethylbenzenthiazoline-6-sulfonic) acid (ABTS) and DPPH free radicals. L. bulgaricus reduced the levels of FBG, 1hBG and 2hBG, and incidence of perinatal complications (P<0.01). Plasma MDA level in the BG group was lower than in the CG group, whereas the levels of SOD, GSH-PX and T-AOC in the BG group were higher than in the CG group (P<0.01). Conclusions: L. bulgaricus improves antioxidant capacity of black garlic in the prevention of GDM.


Introduction
Gestational diabetes mellitus (GDM) refers to abnormal glucose tolerance during pregnancy [1]. GDM is potentially harmful to both mother and offspring, including increased amniotic fluid [

Blood glucose assessment
Oral glucose tolerance test (OGTT) was performed at 24-28 weeks of gestation. The test could be carried out earlier if the women had a high glucose level in urine during their prenatal visits, or had a high risk for GDM [33]. Pregnant women were fasted for 8 h before OGTT to avoid strenuous activity and mental stimulation. The fasting blood was collected at 7 a.m. and FBG was measured using DEXTER-Z II (Bayer Medical Co., Ltd., Leverkusen, Germany). Seventy five grams of glucose solution (200 ml) was taken within 5 min. No food, smoking, coffee and tea were allowed during the trial. Food intake would affect glucose tolerance [34]. Women who smoked had higher 1-h blood glucose (1hBG) than non-smokers, which also affected OGTT [35]. Coffee [36] and tea [37] could improve glucose tolerance and impair the test too. Venous blood was taken after 1 and 2 h after started eating a meal, and 1hBG and 2-h blood glucose (2hBG) after OGTT was measured using DEXTER-Z II.

GDM diagnostic criteria
According to the diagnostic criteria for GDM recommended by the International Association of Diabetes and Pregnancy Group (IADPSG), one of the following criteria was diagnosed as GDM: FBG ≥ 5.1 mmol/l; 75 g OGTT 1hBG ≥ 10.0 mmol/l and 75 g OGTT 2hBG ≥ 8.5 mmol/l [38].

Dietary survey
A 24-h dietary review questionnaire was performed in all pregnant women to investigate dietary intake during the first and the third trimesters. The 24-h dietary questionnaire of the control and the intervention groups was used to calculate the intake of each nutrient according to 'Chinese Food Composition

Inclusion criteria
First prenatal visit was before 12 weeks' gestation. All patients were singleton pregnancy and met the diagnostic criteria for GDW. FBG levels were more than 5.1 mmol/l and/or more than 8.5 mmol/l after 2 h. The patients were informed about the content of the present study and volunteered to participate in the present study.

Exclusion criteria
The patients suffered from hypertension, kidney and or cardiovascular disease. They took the medications that may interfere with sugar and lipid metabolism during pregnancy (e.g. indomethacin, phentolamine, diuretics, phenytoin sodium and pine etc.). Also, the patients taking specified antioxidant were excluded. The patients had placenta previa, threatened abortion and artificial infertility. The patients were lost in the course of the study. Some patients had history of previous adverse pregnancy: unexplained stillbirth or fetus stopped developing.

Trial design
The present study was a parallel randomized controlled trial with double blind parallel designs. All patients were divided into two groups with equal allocation.

Patients grouping
Sample size was determined by using a power test with the expected power of 0.9 and α of 0.5 [39], and required population size of 220 GDM patients with 110 for each group. Of these pregnant women, 324 patients were recruited. GDM experts enrolled all participants. After inclusion and exclusion criteria, 98 patients were excluded, including hypertension (30 cases), kidney disease (8 cases), cardiovascular disease (15 cases), medications that interfered with sugar and lipid metabolism during pregnancy (23 cases), placenta previa (2 cases), threatened abortion (3 cases), artificial infertility (3 cases), lost in the course of the study (6 cases), history of previous adverse pregnancy (3 cases), unexplained stillbirth (2 cases) and fetus stopped developing (3 cases). Finally, 226 patients were selected to participate in the trial (Figure 1). A statistician generated the random allocation sequence by using a random number table created by a computer and the sequence was concealed until interventions were assigned. The present study was parallel randomized controlled trial and the patients were randomly and evenly assigned into the BG and CG groups. The allocation ratio was 1:1 in a trial comparing two different treatments.

Intervention
A third investigator (C.D.) assigned participants to interventions after signed informed consent was obtained from each participant. In the BG group, the patients received 5 g black garlic fermented by L. bulgaricus daily, and in the CG group, the patients received the black garlic without the fermentation with L. bulgaricus daily at home at 11 a.m. in Changchun city (Figure 1). The present study was performed in a double-blind trial, and the investigator and trial personnel, as well as the patients and care providers, were blinded to treatment identity after assignment to interventions. The similar intervention was that black garlic was used in both groups. The treatment duration was 40 weeks from July 2016 to April 2017.

Primary outcome analysis
The primary outcome was the proportion of patients who were screened for GDM with an OGTT within 4 weeks after the study start date. FBG was measured at 7:30 a.m. and 1hBG and 2hBG were measured exactly 1 and 2 h after start of eating a meal on the same day. All blood glucose was measured by using was measured by using DEXTER-Z II (Bayer Medical Co., Ltd., Leverkusen, Germany). The blood glucose was recorded every 4 days.

Secondary outcome analysis
Secondary outcomes were from after 4-week primary outcome analysis to the end of the present experiment. Before the end of the present experiment, pregnancy and fetal perinatal outcomes were investigated: (1) pregnancy outcomes in the women included gestational age at delivery, weight gain during pregnancy, presence of induced labor, cesarean section, and preeclampsia, and measured by pregnancy nursing experts; (2) fetal perinatal outcome included whether there was over-production, stillbirth, neonatal death, low birth weight infants, macrosomia, preterm infants, respiratory distress syndrome, hyperbilirubinemia and/or neonatal intensive care unit (NICU), and measured by perinatal nursing experts.

Measurement of antioxidant activities in GDM patients
Five milliliters of blood was drawn from each patient arm veins with a catheter on the seventh morning of hospitalization within 7-8 a.m. in a stable state. The heparin anticoagulant tube was used to collect 4-5 ml peripheral blood during fasting. The blood sample was centrifuged at 1500 rpm for 10 min. The upper plasma was collected and placed in four centrifuge tubes, and placed in −20 • C refrigerator. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and T-AOC kits were purchased from Jiancheng Co. Ltd. (Nanjing, China). Plasma levels of MDA, SOD, GSH-PX and T-AOC were measured using corresponding kits. MDA, SOD, GSH-PX and T-AOC was measured spectrophotometrically on a UV-Vis spectrophotometer (Hitachi, Japan) at 535, 560, 340 and 520 nm according to manufacturer's instructions, respectively.

Measurement of side effects
The side effects of garlic included unpleasant taste (100%), halitosis (90%) and nausea (30%) [40] and burning sensations [41], but no side effect for black garlic was reported. L. bulgaricus may cause hives, chest tightness, difficulty in breathing, and swelling of face, lips, tongue and/or throat. All these side effects were investigated during the whole period of the present experiment.

Statistical analysis
The data were presented as mean values + − S.D. (standard deviation). Statistical analysis was performed using the SPSS 20.0 statistical software package (IBM-SPSS, Armonk, NY, U.S.A.). A Chi-square test was used to compare count data for primary and secondary outcomes between two groups. The t test (for normal distribution) or Mann-Whitney U test was used to compare measurement data for primary and secondary outcomes between two groups. The statistical difference was significant if P<0.05.

Main ingredients in black garlic
The changes in main ingredients between fresh garlic and black garlic are provided in (Table 1). Before fermentation, the statistical difference for all parameters was insignificant between two groups (

L. bulgaricus increased the contents of glucofuranoside in black garlic
FTIR analysis showed that a significant absorption region from 3150 to 3750 cm −1 for OH stretching frequencies [42] in fresh garlic (Figure 2A,B,D) and an absorption region from 2450 to 3050 cm −1 for CH stretching frequencies [43] in black garlic ( Figure 2C,D). The results suggested that there were some polysaccharides in the black garlic, and also could be found in the fresh garlic. L. bulgaricus promoted the transformation of the polysaccharides in fresh garlic. There was the spectra of glycosides of uronic acids at 1780 cm −1 [44] in fresh garlic (Figure 2A,B). The absorbing values at 1200 and 1100 cm −1 are both assigned to the coupling of CO [45] and CC [46] stretching frequencies ( Figure  2A,B). Absorbance at 930 cm −1 was COH deformation [47] and 900 cm −1 was CH bending region [48] ( Figure 2C,D). The present findings approved that the garlic was with absorbing peaks of polysaccharides. 13 C NMR analysis indicated that there were four peaks in BGP spectra. The peaks at 104.8, 77.5, 77.5, 73.6 and 70.2 ppm were C-1, C-3, C-5, C-2 and C4 in polysaccharides in both the groups before fermentation [49], respectively. After the fermentation, the difference might be caused by the different sugar moiety. Before fermentation, the peaks were 104.0, 76.8, 74.1, 70.6 and 61.8 ppm in both the groups ( Figure 3A,B), which were compatible to β-d-glucopyranoside [50]. After fermentation, the peaks were 110.0, 82.3, 80.6, 75.8, 70.7 and 64.7 ppm in the black garlic fermented with probiotics ( Figure 3C), which was compatible to β-d-glucofuranoside [51], whereas there were the peaks 104.0, 76.8,   Figure 3D). The present findings proved that L. bulgaricus promoted the transformation of the glucopyranoside in fresh garlic to glucofuranoside in black garlic.

L. bulgaricus increased antioxidant capacities of black garlic
Some polysaccharides with strong reducing ability can provide electrons and free radical reactions, which interrupt the free radical reaction chain, and prevent the formation of lipid peroxidation products. Figure 4A showed that the reducing ability of polysaccharides was similar between two groups (P>0.05) and lower than Vc group (P<0.05) before fermentation. The reducing ability of the BGP gradually increased with L. bulgaricus after fermentation and exhibited significant dose-dependent relationship (P<0.05, Figure 4B). T-AOC was similar between two groups (P>0.05) and lower than Vc group (P<0.05, Figure 4C) before fermentation. The T-AOC of the BGP gradually increased with L. bulgaricus after fermentation and exhibited dose-dependent relationship (P<0.05, Figure 4D).
Hydroxyl radicals are very reactive and have strong electron-accepting ability. They can react with many biomacromolecules and are harmful and destructive reactive oxygen free radicals. The hydroxyl radical scavenging ability was similar between two groups (P>0.05) and lower than the Vc group (P<0.05, Figure 4E) before fermentation. The hydroxyl radical scavenging ability of the BGP gradually increased with L. bulgaricus after fermentation and exhibited significant dose-effect relationship (P<0.05, Figure 4F).
The ABTS method is a widely used method for indirect detection of antioxidants. ABTS free radical scavenging ability was similar between two groups (P>0.05) and lower than the Vc group (P<0.05, Figure 4G) before fermentation. ABTS free radical scavenging ability of the BGP gradually increased with the addition of L. bulgaricus after fermentation and exhibited significant dose-effect relationship (P<0.05, Figure 4H).
DPPH is widely used in the evaluation of antioxidants in vitro and a very stable nitrogen-centered free radical. Its ethanol solution is blue-violet and has strong absorption at 517 nm. When an antioxidant is added, blue-violet will change. It is light yellow or colorless, and the absorbance is reduced. DPPH free radical scavenging ability was similar between two groups (P>0.05) and lower than the Vc group (P<0.05, Figure 4I) before fermentation. The scavenging ability of DPPH of the BGP gradually increased with the addition of L. bulgaricus after fermentation and exhibited significant dose-dependent relationship (P<0.05, Figure 4J). Table 2 showed that there were 28 volatile components with higher relative content of garlic in the dormant period, and the highest content of these compounds was diallyl disulfide, allyl formate, diallyl trisulfide, alkene, propyl sulfide and tetrahydro-2-ethylthiophene. There were no significant statistical differences between two groups (P>0.05). Table 3 showed that the main volatile components in fermented black garlic were 29 and 27 kinds of compounds in BG and CG, respectively, and the high-level volatile components were thiane, diallyl disulfide, 2-ethyltetrahydrothiophene, 2-vinyl-1,3 dithiane, N, N -dimethylthiourea etc. In addition to the pungent odor, diallyl trisulfide has burning and sulfur odor, and the irritating odor was significantly reduced after garlic fermentation. The content of thiophene was significantly increased compared with fresh garlic, which produced light fragrance in black garlic. Experiments showed that the irritating odor of garlic was greatly reduced after high temperature fermentation, and the aroma was increased. The total amount of volatile sulfur in black garlic inhibited the synthesis of carcinogenic nitrosamines, the formation and growth of cancer cells, lowered blood pressure and prevented cardiovascular and cerebrovascular diseases. Other low molecular volatile compounds (especially allyl thiol and hydrogen sulfide) subsequently promoted the formation of dipropylene sulfide and cyclic compounds, such as -allyl-thioether and 2-vinyl-1,3-thiane. On the other hand, allyl alcohol also combined with allyl sulfide, alkene propyl mercaptan or alliin/deoxy-aromatic acid to form diallyl sulfide. High-temperature fermentation of garlic significantly improved the pungent odor of fresh garlic and produce a large number of sulfur-containing compounds that were beneficial to the health of GDM patients. They were 2-methyl-1-butanol, linalool, and ethyl acetate in IG group (Table 3). L. bulgaricus increased the aroma smell of black garlic and produced more ingredients.

Clinical characteristics
After the selection of inclusion and exclusion criteria, 226 GDM patients participated in the present experiment and 98 GDM patients were excluded (Figure 1). Among 226 GDM patients, the proportion of pre-pregnancy BMI ≥ 24 kg/m 2 was 43.5%; age ≥ 35 years was 37.6% and PCOS history was 19.5%. The history of large childbirth was 8.8% and the family history of diabetes was 31.9%. There was no significant difference in demographic variables between the control and intervention groups (Table 4, P>0.05).

The comparison of nutrition intake between two groups
Dietary energy [52] and nutrient intake [53] have been reported to be associated with GDM risk or progression. Compared with the control group, the actual intake of nutrients was similar to that of the interventional group, including energy, proteins, carbohydrates, vitamin A, folic acid, calcium, iron and zinc in the early pregnancy (Table 5, P>0.05).

L. bulgaricus could not affect blood glucose levels of primary outcomes
No one withdrew from the present experiment and the GDM number was same for the subjects who were randomly assigned, received intended treatment and analyzed for the primary outcome between two groups. Blood glucose monitoring is a scalable and practical method to prevent GDM progression [54]. After 1-month treatment, FBG, 1hBG and 2hBG levels in the CG group were similar with the BG group (Table 6, P>0.05). FBG, 1hBG and 2hBG difference for the observed coverage of the 95% confidence interval was from 0.25 to 0.73 mmol/l, 0.5 to 1.1 mmol/l and 0.5 to 1.3 mmol/l mg/dl (P>0.05). These results suggested that L. bulgaricus could not affect blood glucose level and glucose tolerance of primary outcomes in a short term.

L. bulgaricus reduced blood glucose levels of secondary outcomes
After 24-and 28-week treatment, no one withdrew from the present experiment. FBG, 1hBG and 2hBG levels in the CG group were higher than in the BG group (Table 7, P<0.01 for 1hBG and P<0.001 for FBG and 2hBG). These results suggested that L. bulgaricus reduced blood glucose level and increased glucose tolerance. FBG, 1hBG and 2hBG difference for the observed coverage of the 95% confidence interval was from 1.5 to 3.3 mmol/l, 1.1 to 2.5 mmol/l and 1.6 to 3.8 mmol/l (P<0.01).

L. bulgaricus improved pregnancy outcomes between two groups
The weight gain of the intervention group was lower than that of the control group during the gestational period from 36.9 to 39.9 weeks (Table 8, P<0.05). Cesarean section and labor induction rate, preeclampsia incidence was not statistically significant between two groups (Table 8, P>0.05). L. bulgaricus improved pregnancy outcomes between two groups by controlling weight gain and weight difference for the observed coverage of the 95% confidence interval that was from 1.65 to 3.89 kg between two groups.

L. bulgaricus reduced the incidence of perinatal complications
The incidence of huge childbirth, premature baby and NICU was lower in the intervention group than that in the control group (Table 9, P<0.05). There was no significant difference for other perinatal complications between the two groups (Table 9, P>0.05).

Comparison of plasma MDA, SOD, T-AOG and GSH-PX between two groups
There was no statistical difference in plasma levels of MDA, SOD, T-AOG and GSH-PX between two groups at admission (Table 10, P>0.05). The results suggested the patients' grouping would not affect the measurement of antioxidant

Side effects
There was no unpleasant taste, halitosis, nausea, burning sensations, garlic odor, hives, chest tightness, difficulty in breathing, and swelling of face, lips, tongue and/or throat caused by black garlic and L. bulgaricus in both groups.

Discussion
L. bulgaricus increased the antioxidant capacities of black garlic by scavenging hydroxyl radicals, ABTS and DPPH free radicals. L. bulgaricus reduced the levels of FBG and 2hBG, and incidence of perinatal complications (P<0.01). There may be other mechanism for the improvement of hyperglycemic state caused by L. bulgaricus. A regular consumption of yogurt with L. bulgaricus can improve human intestinal microbiota [55]. Diabetic patients had an imbalance in gut microbiota [56], which also caused maternal obesity during pregnancy on offspring metabolism, and probiotic interventions could control the risk of obesity and metabolic diseases [57]. The previous review states that the composition of human gut microbiota would affect hyperglycemic states, which are associated with the diabetes with different severities [58]. That until now, no study exists to suggest that gut microbiota affects GDM. Further work is highly needed to explore the underlying mechanisms and accordingly develop therapeutic strategies. Furthermore, L. bulgaricus reduced plasma MDA level and increased SOD, GSH-PX and T-AOC (P<0.01). The present findings demonstrated that L. bulgaricus improved antioxidant activities of GDM patients. L. bulgaricus improved the polysaccharides of black garlic and further prevented GDM progression. In the OGTT test, the levels of FBG were higher in CG group than in BG group after 1 or 2 h of taking sugar (Table 7). The glycemic control effect in the CG group was lower than in the BG group. GDM patients were mainly manifested as postprandial 2-h hyperglycemia [59]. This may be due to the fact that the postprandial insulin secretion of pregnancies with normal glucose metabolism is earlier than that of GDM patients, and glucose is associated with fast recovery in GDM patients [60,61]. After black garlic fermentation with L. bulgaricus, insulin resistance was improved in GDM women. In the control group, the risk of developing GDM increased as the month of pregnancy increased and insulin resistance still worsened. Unlike postprandial blood glucose elevations, FBG elevations are parallel to impaired glucose tolerance. Patients with abnormal FBG are not simply insulin resistant, and may have impaired β-cell damage and insulin secretion.
The effects of black garlic on intestinal microbiome are seldom reported and should be explored in the future work. The extract of black garlic can be used as oral healthcare products and it also has the potential to control oral bacterial infections [62]. The improvement of composition of intestinal microbiota has been reported to reduce blood glucose levels and control body weight gain in the diabetes patients [63]. Improved gut microbes respond to neuroendocrine, and immune biochemical messages, improve hyperglycemic states of host and have health-promoting properties in the therapy of diabetes [58]. Butyrate production of gut microbiota may be one of the important mechanisms in regulating energy metabolism by reducing lipid and glucose levels [64].
Weight gain during pregnancy is a concern for the prevention and treatment of GDM. During pregnancy, body weight gains too fast and fat accumulates, which continuously stimulates the secretion of insulin from pancreatic islet β-cells and triggers hyperinsulinemia. The fatty acid receptors distributed in per unit area of islet cells are relatively reduced, will decrease insulin sensitivity, reduce insulin action, induce excessive secretion of insulin and result in insulin resistance and dysfunction of pancreatic β-cell [65,66]. The present study showed that weight gain in the BG group was lower than the control group (Table 8, P<0.05). The weight gain of pregnant women with early intervention was easily controlled within a reasonable range, suggesting it was necessary for early screening, diagnosis and treatment of GDM. Furthermore, women's dietary intake was studied only in the first and third trimesters but not in the second trimester. Maternal weight change in the first trimester but not the second or the third could affect newborn size [67,68], and thus the first trimester should be selected and the third trimester was selected randomly.
Perinatal period is a critical period of fetal growth and development, and nutrition should be provided for fetal growth and development from the mother. However, long period of maternal hyperglycemic environment will stimulate pancreatic β-cell proliferation, insulin secretion and hyperglycemia on fetus, and cause serious effects on the endocrine system and development of various organs of the fetus [69]. The black garlic fermentation with L. bulgaricus for GDM met maternal and fetal nutritional needs, and achieved glycemic control. Huge children are one of the major adverse outcomes of GDM. Insulin has functions of promoting fat and protein synthesis and inhibiting lipolysis. If the fetus is in a hyperinsulinemic environment, the limbs will develop excessively. Some studies showed that the occurrence of GDM increased not only the incidence of macrosomia, but also the prevalence of obesity in adult offspring [70,71]. Compared with normal weight infants, GDM pregnant women produced more serum cholesterol and triglyceride, so the occurrence of macrosomia and GDM lead to changes in lipid metabolism in neonates [72][73][74]. The present data showed that the incidence of macrosomia and preterm infants was significantly lower in the BG group than that in the CG group (P<0.05).
There were some limitations in the present work. There were many ingredients in black garlic and the molecular mechanism for L. bulgaricus affecting the ingredients of black garlic remains unknown. L. bulgaricus increased the contents of glucofuranoside and reduced the contents of glucopyranoside, which can be used to induce diabetic risk [75] although the effects of glucofuranoside on diabetes remain unclear. L. bulgaricus and black garlic has been used widely in China. However, side effects of black garlic fermentation with L. bulgaricus remain unclear although L. bulgaricus has been used worldwide as functional food. Furthermore, the synergistic effects of the probiotics and black garlic were not explored either. The present study failed to control some confounding variables (obesity and parity) and GDM patients were not stratified based on disease severity. The potential bias may be caused by the selectivity, such as the persons from the same city and garlic was from the same place. Thus, the components of black garlic may vary from different places. Further work is highly demanded to address these important issues.

Conclusions
The content of total sugar, reducing sugar and polysaccharide in black garlic was higher than in fresh garlic. L. bulgaricus promoted the transformation of the glucopyranoside in fresh garlic to glucofuranoside in black garlic. L. bulgaricus increased the antioxidant capacity of the BGP by increasing the abilities of scavenging hydroxyl radicals, ABTS free radicals and DPPH free radicals. After the intervention of black garlic fermentation with L. bulgaricus, the clinical characteristics of GDM were improved by controlling weight gain of GDM patients, avoiding low birth weight babies and macrosomia, and improving birth quality of newborns. L. bulgaricus reduced the levels of FBG, 1hBG and 2hBG, and incidence of perinatal complications. L. bulgaricus reduced MDA level and increased the levels of SOD, GSH-PX and T-AOC levels. L. bulgaricus improves antioxidant capacity of black garlic in the prevention of GDM.