The sourdough fermented wheat germ was characterized based on microbiological, physico-chemical and biochemical features. During incubation, the release of the non-glycosylated and physiologically active 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone was almost completed during 24 h. Compared to the control, the concentration of the above bioactive compounds increased almost 4 and 6-folds. Both raw wheat germ (control) and sourdough fermented wheat germ were ex vivo assayed for the anti-proliferative activity towards various cell lines of germ cell tumor, colon carcinoma and ovarian carcinoma. While no effect was found for the raw wheat germ, the sourdough fermented preparation markedly and variously affected the human tumor cell lines. The values of IC50 ranged from 0.105 ± 0.005 to 0.556 ± 0.071 mg/ml, with a median value of IC50 of 0.302 mg/ml. Conclusions: These results are comparable to those found for other well-known pharmaceutical preparations, and may disclose the use of the sourdough fermented wheat germ as an ingredient, nutritional supplement and/or anticancer drug.

Background Wheat germ, corresponding to 2-3% of the total weight of wheat kernel, is almost systematically removed during milling since it adversely affects the shelf-life and the processing quality of the flour [1]. Due to the high concentration of functional compounds, wheat germ is one of the most attractive and promising source of α-tocopherol, vitamins of group B, dietary fiber, polyunsatured fats, minerals and phytochemicals [1]. Usually, the effects of wheat bran on human physiology are grouped into nutritional, mechanical (mainly due to the fiber content) and antioxidant (mainly due to phenolic acid and alkylresorcinols) [2]. Besides the positive effects, the use of wheat germ in bakery industry is still challenging because of the poor stability and the presence of anti-nutritional factors such as: (i) raffinose which is not digested by pancreatic enzymes but metabolized by gas-producing bacteria of the large intestine, thus causing disorders such as flatulence [1]; (ii) phytic acid which markedly decreases the mineral bioavailability [3]; and (iii) wheat germ agglutinin (WGA) which is responsible for the hyperplastic and hypertrophic growth of the small bowel and pancreas [4]. Treatments by heat, microwave and extrusion [4] or the addition of antioxidants [5] were considered to increase stability of wheat germ. Despite their effectiveness, these technology approaches are in some cases expensive, not completely resolving, and somewhat decreasing the nutritional value of wheat germ [5]. Recently, some studies exploited the potential of microbial enzymes or sourdough fermentation to process wheat germ and to enhance the nutritional and sensory properties of related cereal based foods [1].

 Lactic acid bacteria isolated from wheat germ were selected based on technology features and used as starters for the manufacture of fermented wheat germ through a biotechnology, which resembled the traditional sourdough fermentation [1]. Sourdough fermentation is one of the oldest food biotechnologies, which was extensively studied for the effects on the sensory, structural, nutritional and shelf life properties of leavened baked goods [6]. Acidification, proteolysis and activation of a number of enzymes as well as the synthesis of microbial metabolites cause several changes during sourdough fermentation, which affect the dough and baked good matrix and, in turn, influence the nutritional/functional quality [6]. Overall, sourdough fermentation stabilized and enhanced the nutritional properties of wheat germ. Besides, lactic acidification markedly decreased the lipase activity of the sourdough fermented wheat germ compared to the levels found in the raw wheat germ [1]. In the last decade, several studies described the promising cytotoxic activity of a yeast (Saccharomyces cerevisiae) fermented wheat germ extract (FWGE ®) towards cancer cell lines and during in vivo clinical trials [7,8]. Recent data suggested that the antiproliferative, antimetastatic and immunological effects of this preparation are mainly attributed to two quinones, 2- methoxy benzoquinone (2-MBQ), and 2,6-dimethoxybenzoquinone (2,6-DMBQ) [7]. This study aimed at investigating the effect of the sourdough lactic acid bacteria fermentation on the release of 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone, which are naturally present in the wheat germ as glycosylated and non-physiologically active form.

 The cytotoxic activity of the sourdough fermented wheat germ was determined through ex vivo assays on several human cancer cell lines. Results β-glucosidase activity Preliminarily, the β-glucosidase activity of the forty strains of lactic acid bacteria, previously isolated from wheat germ, was assayed on the synthetic substrate pNPG. Figure 1 summarizes the results. The enzyme activity varied within a wide range: from 0.011 ± 0.005 to 0.269 ± 0.014 U. The median value of the distribution was found to be 0.06 U, and the values corresponding to the 25th and 75th percentile of the data were 0.014 and 0.130 U, respectively. In particular, the well-characterized strains [1,9] Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 showed the highest β-glucosidase activity (0.269 ± 0.014 U) and a value located in the non-outlier range (higher than the 75th percentile of the data, 0.140 ± 0.008 U), respectively. Besides L. plantarum LB1 and L. rossiae LB5, Weissella confusa G8 (0.062 ± 0.005 U), and Pediococcus pentosaceus G1 (0.015 ± 0.004 U), representative of the strains having β-glucosidase activity close to the median value, and of the strains having low enzymatic activity, respectively, were used for wheat germ fermentation with the aim of investigating the correlation with the benzoquinones release. Nevertheless, based on this preliminary assay and since both L. plantarum LB1 and L. rossiae LB5 were previously selected based on the kinetic of acidification and other technology features [1,9], these two strains were further used in combination to ferment wheat germ. 

Sourdough fermentation of wheat germ The averaged values of raw wheat germ (RWG) were as follows: moisture 11.08 ± 0.22%, protein (N x 5.70) 28.50 ± 0.72% of dry matter (d.m.); fat 7.95 ± 0.06% of d.m.; and ash 3.82 ± 0.09% of d.m. RWG had pH of 6.36 ± 0.05 and Total titratable acidity (TTA) of 18.3 ± 0.11 ml of 0.1 M NaOH/10 g. The concentration of total free amino acids was 15614 ± 134 mg/kg. As estimated by plate count, cell density for yeasts and presumptive lactic acid bacteria in RWG was 1.5 ± 0.5 x 103 and 2.0 ± 0.5 x 105 cfu/g, respectively. After 24 h of incubation at 30°C, the cell density increased to 5.5 ± 1.5 x 104 and 4.2 ± 0.7 x 107 cfu/g, respectively, and the pH decreased to 5.51 ± 0.07. The persistence and the dominance of L. plantarum LB1 and L. rossiae LB5 during fermentation of SFWG was confirmed through RAPD-PCR analysis. As estimated by plate count and the use of RAPD-PCR, cell densities at the end of fermentation were 6.5 ± 0.4 x 109 and 2.3 ± 0.5 x 109 cfu/g for L. plantarum LB1 and L. rossiae LB5, respectively. The pH of the sourdough fermented wheat germ (SFWG) was 4.16 ± 0.03. Compared to RWG, TTA increased to 24.9 ± 0.07 ml of 0.1 M NaOH/10 g. SFWG was further characterized for features previously recognized as related to nutritional and technology quality [1,9]. The water/salt-soluble extracts of RWG and SFWG were used to determine the lipase activity. The minimum concentration of the crude enzyme extract that failed to give a detectable zone of hydrolysis was, respectively, 50.2 ± 1.4 and 163.8 ± 1.6 μg/ml.

 After fermentation, the concentration of total free amino acids increased to 23491 ± 94 mg/kg. Almost all the free amino acids increased. Leu, Lys, Phe, Val, His, Ala, and Met showed the highest increase, which varied from 2 (Ala) to ca 10-fold (Leu and Met) compared to the levels found in RWG. Apart from the increase during fermentation, Arg, Ser, GABA and Lys were the free amino acids found at highest concentration in SFWG (4070 ± 21, 2252 ± 14, 2031 ± 18 and 1944 ± 27 mg/kg, respectively). Lactic and acetic acids were not detectable in RWG. After fermentation, 0.98 ± 0.02 and 0.25 ± 0.02% of lactic and acetic acids were, respectively, found in SFWG. The methanol extracts of RWG and SFWG contained 0.49 ± 0.02 and 0.65 ± 0.02 mM of total phenols (expressed as gallic acid equivalent), respectively. Release of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone during sourdough fermentation As determined by selective extraction and HPLC analysis, RWG contained 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone at the concentrations of 0.082 ± 0.01 and 0.035 ± 0.005 mg/g, respectively. 

After sourdough fermentation with L. plantarum LB1 and L. rossiae LB5 [1], the concentrations of the above two quinones increased to 0.415 ± 0.016 and 0.252 ± 0.013 mg/g, respectively. Compared to SFWG obtained by the inoculum of the two starters together, the concentration of both benzoquinones in wheat germ fermented with the lactic acid bacteria singly was ca. 70, 40, 10%, and not detectable for L. plantarum LB1, L. rossiae LB5, W. confusa G8, and P. pentosaceus G1, respectively. Aiming at describing the kinetic of release, aliquots of SFWG were analyzed at different intervals of time during 72 h of incubation at 30°C. Further incubation of RWG (control), did not allow a significant (P > 0.05) increase of the concentration of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone (Figure 2). Nevertheless, a significant (P < 0.05) decrease of the value of pH was found (pH 5.02 ± 0.13) under these conditions. A first significant (P < 0.05) increase of the concentration of 2- methoxy benzoquinone and 2,6-dimethoxybenzoquinone which corresponded to 102 and 138%, respectively, was already found after 2 h of incubation of SFWG (Figure 2). The concentrations of the two quinones further increased until 24 h, observing the highest increment between 12 and 18 h of incubation. After 48 h of incubation, SFWG contained 2-methoxy benzoquinone and 2,6- dimethoxybenzoquinone at the levels of 0.415 ± 0.016 and 0.252 ± 0.013 mg/g, respectively.

 Nevertheless, these small increases corresponded to a decay of the organoleptic characteristics of the wheat germ (data not shown), that become too acidic to taste. Not significant (P > 0.05) increases of the concentrations of the two quinones were found during late incubation. Anti-proliferative activity of SFWG in human cancer cell lines The anti-proliferative activity during 96 h of continuous exposure to SFWG was determined on different human tumor cell lines, using the SRB assay. The values of IC50 were calculated using Sigma Plot and the collected data are summarized in Figure 3. The values of IC50 of SFWG ranged from 0.105 ± 0.005 to 0.556 ± 0.071 mg/ml, with a median value of IC50 of 0.302 mg/ml. The highest antiproliferative activity was found on ovarian carcinoma cells A2780, while the lowest effect was observed on colorectal carcinoma cells HT-29, followed by HCT-8. The values of IC50 for germ cell tumor cells 1411HP and H12.1, and colorectal carcinoma cells DLD-1 were intermediate (0.198 ± 0.071 to 0.344 ± 0.044 mg/ml). No anti-proliferative activity was found for RWG in the range of concentration used. Discussion Quinones consist of a class of bioactive compounds with promising potential as components for anticancer chemoteraphy drugs [11]. Concerning vegetable foods, wheat germ is probably the best reservoir of the glycosylated and non-active forms of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone. The non-glycosylated forms of both these compounds have antimicrobial and immune-stimulatory effects [12,13]. In particular, 2,6- dimethoxy-1,4-benzoquinone, which was identified in a large variety of plant families in addition to wheat [14-16], exerts a strong in vitro cytotoxicity against human tumor cell lines [13,17,18].

 The conversion of glycosylated to non-glycosylated forms requires β-glucosidase activity, which allows a remarkable increase of the functional activities, including the proven anticancer effect [12]. The use of Lactobacillus zeae and Pichia pijperi as starters for wheat germ fermentation promoted the release of the non-glycosylated forms of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone during 48 h of incubation [12]. A fermented and well standardized extract of wheat germ (trade-name FWGE ®) was extensively studied for treatment of cancer and autoimmune diseases [19-21]. FWGE ® is made through fermentation of wheat germ with baker’s yeast (Saccharomyces cerevisiae), which harbors β-glucosidase activity. The aglycone-type 2- methoxy benzoquinone and 2,6-dimethoxybenzoquinone were recognized as the most important bioactive compounds of this preparation [13,21]. The protocol for the manufacture of FWGE ® includes the use of a water-soluble extract of wheat germ, the fermentation of this extract, and further concentration and drying. FWGE ® induced apoptosis in many cancer cell types, including leukemia, melanoma, breast, colon testicular, head and neck, cervical, ovarian, gastric, thyroid, and brain carcinomas [7,22,23]. 

The active compounds contained in the fermented wheat germ interfere with anaerobic glycolysis, pentose cycle, and ribonucleotide reductase. Tumor cells were killed by the induction of apoptosis via the caspase-poly [ADP-ribose] polymerase-pathway, interacting with different anticancer drugs [8]. Like S. cerevisiae, lactic acid bacteria are food-grade microorganisms largely used for the manufacture of a variety of fermented foods. β-Glucosidase activity is widespread within lactic acid bacteria, even though the level of expression is dependent on the strain [24]. βGlucosidase activity of lactic acid bacteria was already successfully used to release equol and bioactive isoflavone aglycones from soymilk [24,25]. Lactic acid bacteria isolated from wheat germ were characterized and selected based on technology performance to be used as starters for fermenting wheat germ [1]. Fermentation with selected and autochthonous lactic acid bacteria allowed a long storage of wheat germ, mainly due to lipase inactivation [1,9]