Abstract

Interactions between lactic acid bacteria (LAB) and dietary polyphenols are not fully understood. This study determined the prebiotic effects of grape seed flour (GSF, a byproduct of wine making) and their polyphenol extracts (GSE) on the growth of 10 strains of probiotic LAB recently isolated from the kefir and pathogenic Clostridium perfringens. Growth rate of the LAB, Leuconostoc mesenteroides DH 1608, was the highest among 10 strains of LAB when cultivated with GSF. GSE, containing an equivalent amount of polyphenol in GSF, also promoted the growth of L. mesenteroides DH 1608 but at a lower rate compared to GSF. Growth of C. perfringens decreased significantly at 10 mg/mL of GSF, but GSE did not affect the growth. In conclusion, GSF may be a useful prebiotic dietary supplement due to its selective antimicrobial capacity via stimulation of probiotic bacteria and inhibition of pathogenic bacteria. A symbiotic combination of selected lactic acid bacteria and GSF may improve gut health.

1. Introduction

In recent years, there has been growing interest in wine grape seed flour (GSF) which is a by-product of wine making due to its potential health beneficial effects on obesity, cardiovascular disease, nonalcoholic fatty liver disease (NAFLD) ^{1, 2}. Interestingly, these effects were closely associated with regulation of intestinal microbiota suggesting a possible prebiotic role of GSF ². GSF contains two-thirds of the extractable flavonoids of grape with high amounts of flavonoids, flavan-3-ols (flavanols) such as catechin, epicatechin, their 3-O-gallates, and (epi)catechin dimers, oligomers, and polymers ³. The oligo- and polymeric catchins are not readily absorbed by the small intestine and reach the colon where they are metabolized by gut bacteria ⁴. Intensive studies have recently explored interactions between aqueous or alcoholic extractable polyphenols and intestinal microbiota. Extractable polyphenols from grape seed pomace stimulated growth of Lactobacillus acidophilus and Bifidobacterium adolescentis and B. bifidum ⁵ and inhibited the growth of pathogenic bacteria such as Clostridia ^{6, 7, 8}. However, these studies have not evaluated the effects of whole wine grape seed that is high in dietary fiber and other bioactive components as well as polyphenols, on microbial growth selectively.

Kefir, a probiotic, is a traditional fermented milk of Caucasus Mountains where it is famous for increasing longevity. In addition to lactic acid bacteria, it consists of yeast and acetic acid bacteria that comprise together approximately 10⁹ CFU/mL. It has shown potential health benefits including anti-obesity, anti-diabetic, anti-stress, hypocholesterolemic, and antimicrobial activity ^{9, 10}. Previously, we isolated and characterized major lactic acid bacteria from Kefir. These lactic acid bacteria were shown to have a high in-vitro survival rate at low pH, were able to grow in an artificial intestinal environment, were bile acid tolerant and had an anti-obesity effect indicating roles as a probiotic ^{11, 12}.

To the best of our knowledge, there has been no study reporting the selective modulation of growth of lactic acid bacteria and pathogenic bacteria by whole wine grape flour. This study aimed to determine whether polyphenol-rich whole GSF stimulates the growth of lactic acid bacteria isolated from probiotic Kefir and inhibits the growth of toxigenic bacteria (Clostridium perfringens) in in-vitro system and compares these effects with GSE.

2. Materials and Methods

Lactococcus lactics subsp. Lactics (DH 1601~1603, 1605), Leuconostoc mesenteroides (DH 1604, 1606~1609) and Lactobacillus kefiri DH5 isolated from kefir fermented milk (Konkuk University, Korea) were used. Clostridium perfringens (KCTC 3269) were cultured in Reinforced Clostridial Broth (MBcell, Seoul Korea). Skimmed milk powder for the lactic acid bacteria stock was purchased from Sigma-Aldrich Chemical Co. Ltd (St. Louis, MO, USA). Lactic acid bacteria were incubated in Man-Rogosa-Sharpe (MRS) broth (Difco, Detroit, MI, USA). MRS agar plates were made by adding agar powder. Chardonnay grape seed flour (GSF) was purchased from Sonomaceuticals, LLC/WholeVine Products (Santa Rosa, CA, USA). Dimethyl sulfoxide (DMSO) was purchased Daejung, Korea.

Wine grape seed flour (0.3 g) was mixed with 18 mL of 80% methanol or water, sonicated (SD-350H, Seong Dong, Seoul, Korea) for 30 min. The suspension was filtered through filter paper (Whatman No. 1, Whatman International Ltd., Maidstone, England), and the residue was re-extracted with 80% methanol or water for 30 min. The filtrate was freeze dried (Freeze dryer, FD5508, Ilshin Lab Co., Ltd., Dongducheon, Korea). The dried methanol extract (SGME) and water extract (SGWE) were stored at -20°C for further analysis.

GSF (5 and 10 mg), SGME, SGWE, or catechins were individually added into 3 mL of MRS broth containing 0.1% dimethyl sulfoxide (DMSO) and sonicated for a total of 5 min; 50 sec operate time and 10 sec interval time, repeated 5 times. Then 10⁷ CFU of lactic acid bacteria were inoculated and anaerobically cultured at 30°C for 48 hrs. Number of L. lactics subsp. Lactics and L. mesenteroides were counted after 24 h, 48 h. L. kefiri DH5, was enumerated after 96 h. C. perfringens was anaerobically incubated in Reinforced Clostridial broth at 37°C for 12 h to determine CFU.

The microbial growth was determined as the growth with GSF compared to without GSF by calculation [number of bacteria (log CFU/ml) in the presence of 5 mg GSF – number of bacteria (log CFU/ml) in 0 mg of GSF] or [number of bacteria (log CFU/ml) in the presence of 10 mg GSF – number of bacteria in 0 mg of GSF].

To apply the same amount of catechins across the samples, the catechin content of SGWE and SGME was determined in an equal initial amount of GSF based on extraction yield (SGWE 8.2% and SGME 5.7%). SGWE1 and SGME1 contain same amount of extract as 5 mg of GSF before extraction. SGWE2 and SGME2 contain same amount of extract as 10 mg of GSF before extraction.

0.2 g of GSF was extracted with 20 mL methanol for 30 min with shaking. After 10 min of sonication, the mixture was centrifuged and supernatant was analyzed for flavonoids by HPLC and total phenolics. Total phenolic content was determined using the Folin-Ciocalteu method ¹³ and flavonoid contents were analyzed using a standard HPLC method ¹⁴.

All data were expressed as mean ± SEM and statistical analysis was carried out using SPSS (Version 21.0, SPSS INC., Chicago, IL, USA)

3. Results and Discussion

Ten strains of lactic acid bacteria (Lactococcus lactics subsp. Lactics, Leuconostoc. mesenteroides, and Lactobacillus kefiri) that we isolated recently from kefir were cultured with GSF for 48 hrs and the strains that grew best were selected for further study. L. mesenteroides DH 1608 showed the best growth in the presence of both 5 mg and 10 mg of GSF (Table 1). The relative growth of most of the kefir derived lactic acid bacteria was greater in 5 and 10 mg of GSF than in broth without GSF (Table 1).

Growth stimulation by GSF on lactic acid bacteria may be due to substrate availability since lactic acid bacteria are able to utilize phenolic compounds (high in GSF) ⁶. GSF also contains about 60% dietary fiber that are fermentable. Several components of polyphenols (especially p-coumaric acid, epicatechin and catechin) improved the growth of Lactobacillus rhamnosus ⁷. Consistently, increased growth of lactic acid bacteria was associated with the amount of catechins in GSF 5 and 10 mg/mL, 35 and 70 μg, respectively. Enzyme activities in lactic acid bacteria played a critical role in utilization of polyphenol components in grape seed extract ¹⁵. Therefore, L. mesenteroides DH1608 may have enzymes able to efficiently metabolize polyphenols such as catechins. Further research is necessary to determine if polyphenol metabolism is the limiting factor for growth.

Because Leuconostoc mesenteroides DH 1608 grew best in the presence of GSF, its growth in grape seed flour extracts (SGWE, SGME) and catechins were examined. After 48 h incubation, growth of L. mesenteroides was greatest with 5 and 10 mg of GSF compared to groups containing GSF extracts, SGWE and SGME (Figure 1). 5 mg of GSF increased the growth by 28% and 15% as compared to SGWE1 and SGME1, respectively. 10 mg of GSF increased the growth by 32% and 16%, in comparison with SGWE2 and SGME2, respectively (Figure 1). These results suggest that growth of L. mesenteroides DH 1608 is optimized by the presence of insoluble components, likely dietary fiber, of grape seed flour compared to the grape seed extracts. Previously, growth stimulation of Lactobacillus acidophilus was observed with grape pomace extracts ⁶. However, the present study demonstrated that the growth of lactic acid bacteria derived from kefir was greater with whole grape seed flour than with grape seed extracts. This result indicates that whole GSF may have additional bioactive components (such as dietary fiber) that contribute to growth stimulation. Soluble dietary fiber has been reported to stimulate growth of lactic acid bacteria from kefir ¹⁶. The growth of L. mesenteroides was not observed with catechins 3, 5, 10 mg. However, 2 mg of catechins greatly stimulated its growth and may have contributed to growth of some lactic acid bacteria in this study (data not shown). A previous study ¹⁷ also reported that greater than 3 mg of catechins is likely to suppress the growth of lactic acid bacteria.

After 12 h incubation anaerobically, the growth of C. perfringens was inhibited only by 10 mg of GSF (Figure 2); 25% in comparison with SGWE2 and 25% compared to 5 mg GSF. Polyphenols such as catechin and epigallocatechin gallate have been reported to control biofilm formation of pathogenic bacteria ¹⁸. Gram-positive pathogens are 8~16 times more sensitive to epigallocatechin gallate (EGCG) than gram-negative pathogens ¹⁹. Interestingly, the current study showed that C. perfringens, gram-positive enteropathogen was more sensitive to GSF than GSE (extracts), indicating whole GSF may have bioactive components in addition to polyphenolics that are more effective for inhibition of C. perfringens growth. Furthermore, 35 μg or 70 μg of catechins in both SGWE1&2 and SGME1&2 was not enough to inhibit the growth of C. perfringens. This study shows that Chardonnay grape seed flour selectively inhibits a harmful pathogenic bacteria more effectively than grape seed flour extracts.

Change in pH is indicator of growth of bacteria. The pH of the culture medium was determined at 48 hours after incubation with L. mesenteroides DH 1608. The pH was 4.35 and similar to control, GSF 5 mg and 10 mg, SGWE1, SGWE2, SGME1, and SGME2 (Fig 3A). However, the pH of L. mesenteroides DH 1608 cultured with catechins was higher than GSF and GSE. Lactic acid fermentation lowers the pH. The lower pH indicates active lactic acid fermentation in the presence of GSF and GSE groups. The higher pH in catechins 3, 5, 10 mg indicates growth inhibition of L. mesenteroides DH 1608 by catechins.

The pH of media 12 h after incubation with C. perfringens was highest with GSF 10 mg (pH 6.35) among all groups (pH is about 4.65) (Figure 3B), indicating growth of C. perfringens was inhibited by GSF 10 mg.

In summary, GSF stimulated growth of most lactic acid bacteria isolated from kefir. Growth stimulation of L. mesenteroides DH 1608 was highest among the lactic acid bacteria isolated from kefir. When compared to GSE (SGWE and SGME), GSF revealed greater capacity to stimulate the growth of L. mesenteroides DH 1608. In contrast, GSF inhibited the growth of C. perfringens whereas GSE did not affect growth of this toxigenic bacteria.

We demonstrated that selective growth stimulation of probiotic lactic acid bacteria derived from kefir and growth inhibition of pathogenic C. perfringens by Chardonnay grape seed flour was greater than grape seed extracts. The symbiotic combination of selected lactic acid bacteria from kefir and GSF may provide health promotion via synergism of probiotic growth in the gut.

Acknowledgments

This study was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2018R1A2B6001097).

Conflict of Interest

The authors have no conflicts of interest to declare.

References