Stabilizing Effect of Vitamin C on Sulforaphane Level in A Controlled-Fermented Broccoli Sprouts

Abbreviations

LAB: Lactic acid bacteria

HPLC: High-performance liquid chromatography

MRS-agar (broth): de Man, Rogosa and Sharp-agar

B: Bifidobacterium

ITCs: Isothiocyanates

HoLAB: Homofermentative Lactic Acid Bacteria

HeLAB: Heterofermentative Lactic Acid Bacteria

Introduction

Sulforaphane is known as a long-lasting antioxidant, who’s anti-cancerogenic effects were confirmed by in-vitro studies conducted by John Hopkins University School of Medicine [1-11] and confirmed by multiple publications [12-14]. Sulforaphane is obtained from cruciferous vegetables (the family of Brassicaceae, formerly Cruciferae), non-limiting examples of which include brussels sprouts, cabbages, cauliflower, broccoli particularly broccoli sprouts). A series of encyclopedias have extensively reported on cruciferous vegetable origin, genetics, and chemistry, especially how glucoraphanin (a glucosinolate) reacts with myrosinase (an enzyme present in the plant cell) to form sulforaphane [15-19,11]. The protective effect of these micronutrients is due to the inhibition of phase-I carcinogen- bioactivating enzymes and induction of phase-II detoxifying enzymes [5-10]. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite formed from glucoraphanin by the enzyme myrosinase [3-5,20,21]. The reaction between myrosinase and glucosinolate occurs in the gut after glucosinolate has been made available by chewing such that sulforaphane is produced after the swallowing event occurs.

Storage, processing, and cooking can change ITCs formation from glucosinolate and affect the cruciferous vegetables' anticancer activity [12]. Intake of raw cruciferous vegetables provides two to nine times the number of ITCs in humans compared with similar intakes of their cooked counterparts due to heat-inactivating myrosinase, which reduces sulforaphane formation [13,14,22,23].

Human intestinal microflora also possesses myrosinase activity, able to partially hydrolyze ingested glucosinolates which is an advantage to this study with the use of Lactobacilli strains. Gut microflora is mainly responsible for converting ingested glucosinolates to their cognate isothiocyanates [14].

Despite their nutritional and health benefits, including being a source of sulforaphane, broccoli sprouts have certain disadvantages that have limited their use. For instance, broccoli sprouts tend to spoil quickly, and contamination with coliform bacteria is a well-known safety issue, resulting in unwanted additives often accompanying broccoli sprouts. Several studies have elucidated microbial fermentation's positive impact on product shelf life and the protective properties of lactic acid fermentation. Balows et al., 1991 also highlighted the prokaryotic ecophysiology, characterization, and applications [24-46].

Apart from providing an edible fermented new product capable of delivering sulforaphane's nutritional benefits (figure 1), this study offers ways to avoid drawbacks associated with the consumption of cruciferous vegetables, especially broccoli sprouts.

Materials and Methods

Processing Brief Description'

Figure 2: represents a flow diagram of a process for probiotics broccoli sprouts.

As outlined in figure 2, we sanitize broccoli seeds before being planted to grow broccoli sprouts. As used herein, broccoli sprouts will refer to new growth from seed germination, a plant that is a few (e.g., three to four) days old. After harvest, the sprouts are washed in purified water and dried to reduce the presence of bacteria on the sprouts. The sprouts are then combined with LAB strains and ingredients (<1% NaCl) which trigger a lactic acid fermentation that transforms glucosinolate contained in the sprouts into sulforaphane-containing product. Fermentation produces sulforaphane without requiring chewing or physically breaking up the sprouts to make the glucosinolate available and without the requirement for contacting and reacting the sprouts with myrosinase. The broccoli sprouts are combined with twelve strains of Lactobacilli, which are part of the lactic acid bacteria (LAB) group and known as probiotics. The novelty of this study is the addition of vitamin C at the earlier stage (5-10 minutes) during LAB growth lag phase.

The fermentation has been successfully conducted in fermenters under anaerobic and dark environments (i.e., shielded from and without natural and artificial lighting) at room temperatures of about 20oC.

Labs and Methods

In this particular study, organic broccoli sprouts germinated over three days were washed three times in non-chlorinated purified water, after which the following twelve selected strains of lactobacilli were added: B. bifidum, B. lactis, B. infantis, B. longum, L. acidophilus, L. brevis, L. bulgaricus, L. paracasei, L. plantarum, L. rhamnosus, L. salvaricus, and Streptococcus thermophilis from “Nature” were used as starter cultures.

These chosen strains are known for their physiological benefits and antimicrobial capabilities. Sea salt <1% and, especially, vitamin C added to this mixture create an early acidic pH suitable for lactic acid fermentation/Reference samples without vitamin C, conducted anaerobically in a dark environment at about 200C for seven days in handmade 3-layered wood-ceramic-glass fermenter (figure 3).

LAB-Analyses: By CMMEF, 4th Ed.; MRS-Agar (broth); E. coli & Coliform (by Petrifilm-AOAC 991.14)

Sulforaphane Content Evaluation: By Solid Phase Extraction (SPE)-High-Performance Liquid Chromatography (HPLC) by Han D.et al.

Minerals and Vitamins Evaluation: Calcium by ICP (AOAC 984.27); Iron by ICP; Vitamin A-Beta Carotene (Analyst 1984 109:48), Vitamin C (HPLC)- 53 mg Vitamin C in 113 g sprouts

Figure 3: Three- layered Fermenter Technology (external layer:woody#2, middle layer: ceramic#4, internal layer: glass#1;door opening#3; reduced air circulation #5)

Results

The process from this study yields a microbiologically safe and durable natural formula that contains sulforaphane before being ingested by an individual and is capable of delivering benefits to the individual when ingested.

After seven days at these conditions described above, the mixture was found to be fermented, and the mixture tested positive (0.130 mg/g) for sulforaphane. The Lactic acid bacteria (LAB) count of the mixture remained high (2500000 cells/g), pH-decrease from 5.0 to 3.9 (Figure 4) and no coliform bacteria was detected. Following storage of the fermented mixture in a refrigerator over a two-month period, coliform bacteria were not detected either. After six months of storage in a refrigerator, the amount of sulforaphane dropped to 50% (0.0651 mg/g), which is a good result given the instability of that phytochemical.

As such, this study processing method results in a broccoli sprouts mixture containing sulforaphane-probiotics with notable nutritional benefits, enriched with vitamins and minerals, which contribute to answering world challenges regarding food scarcity and sustainability.

                                                   Table 1: Nutritional and microbiological evaluation of fermented broccoli sprouts

Figure 4: pH-Decrease within lag-phase corrreponding to LAB Increase (log cell count/ml)

Conclusion and Discussion

The broccoli sprouts combined with twelve specific LAB, as described in this study yielding to the current new product, have been found to trigger a lactic acid fermentation that transforms glucosinolate contained in the sprouts to sulforaphane. Selected Lactobacilli (HeLAB and HoLAB) strains employed by the process in this study yield a sulforaphane-containing product and enhance the stable sulforaphane levels [13,14,22,23].

Lactic acid Fermentation from this study produces sulforaphane without requiring chewing or otherwise physically breaking up the sprouts to make the glucosinolate available and without the requirement for contacting and reacting the sprouts with myrosinase outside the human GI (Gastrointestinal)-tract.

The end product from this study can remain in the form of a consumer food item that can be directly ingested by an individual.

In addition to consumer food items, this study fermented products may be processed and packaged for use by medical personnel and nutritionists, in a variety of industries and practices, for example, as a pill, powder, for use in pharmaceuticals, nutraceuticals, preventive medicines, food processors, etc. This fermented product can particularly benefit individuals with digestive or other specific health issues. The shelf-life of this fermented product is up to two mounts between 10oC to 15oC temperature.

While this study has focused on twelve LAB strains, new studies could use other forms of the mixture; for example, though twelve specific strains of Lactobacillus were used in this study, hypothetically, it may be possible to omit or add some of the used strains of lactobacilli, and yet yield a fermentation that may produce a suitable fermented mixture that contains a sustainable amount of sulforaphane. In addition, it is foreseeable that multiple cruciferous vegetables could also be combined and fermented. We should keep in mind that the processing of Brassica vegetables has negatively impacted the contents of glucosinolates and has reduced their health-protective capabilities [12]. The other question remains on the impact of the ratio of homo- versus heterofermentative strains on the stabilization of sulforaphane level. On the contrary, it is fair to affirm that this study’s vitamin C addition at the lag phase in three-layered fermenter is the turning and essential point of this research paper’s contribution. It brings some hope regarding the end product’s sulforaphane levels and stability, which may contribute positively to food sustainability challenges and opportunities. You can grow it in your environmentally friendly backyard, and its scope provides countless applications [5-10]. Even though the daily value for sulforaphane is still unknown, its amount in this paper’s results is encouraging for consumers with oro-maxillofacial chewing issues. One could investigate monthly radiological and imaging evaluations of sulforaphane using this study’s new processing method.

Acknowledge

This study’s HPLC, minerals, and vitamin testing were supported by the Merieux Laboratories, USA.

Author & Co-Author Contributions

The manuscript was conceived, conducted and written by the author and reviewed by the co-author. Both agreed on the last draft.

Conflict of interest

The author declares no conflict of interest.

References