3.1 Search protocol
The search protocol for the literature review is presented in Figure 1. First search was conducted using PubMed-database to investigate, what is known about in vitro protein digestibility of wheat, barley and faba bean. A following search statement was used:
((in vitro protein digest*[Title/Abstract] OR simulated protein digest*[Title/Abstract] OR in vitro gastrointest*[Title/Abstract] OR simulated gastrointest*[Title/Abstract])) AND (wheat[Title/Abstract] OR Triticum aestivum[Title/Abstract] OR barley[Title/Abstract] OR hordeum vulgare[Title/Abstract] OR faba bean[Title/Abstract] OR fava bean[Title/Abstract] OR broad bean[Title/Abstract] OR vicia faba[Title/Abstract])
The second search was conducted using PubMed-database to investigate the impact of germination on protein digestibility of cereals and legumes with a following search statement:
(((cereal* OR grain* OR legume* OR pulse* OR wheat[Title/Abstract] OR triticum aestivum[Title/Abstract] OR barley[Title/Abstract] OR hordeum vulgare[Title/Abstract] OR faba bean[Title/Abstract] OR fava bean[Title/Abstract] OR broad bean[Title/Abstract] OR vicia faba[Title/Abstract])) AND (malt[Title/Abstract]
OR malte*[Title/Abstract] OR malti*[Title/Abstract] OR germin*[Title/Abstract] OR sprout*[Title/Abstract])) AND protein[Title/Abstract] AND digest*[Title/Abstract]
The second PubMed-search was complemented with similar search using Web of Science -database but the search result was limited to studies published from the year 2000 to present day:
TOPIC: (cereal* OR grain* OR legume* OR pulse* OR wheat OR triticum aestivum OR barley OR hordeum vulgare OR faba bean OR fava bean OR broad bean OR vicia faba) AND TOPIC: (in vitro protein digest*) AND TOPIC: (malt OR malte* OR malti OR germin* OR sprout*)
Additionally, several articles were handpicked from the references of relevant articles. Articles without access to full text in English were excluded.
Figure 1. Search protocol.
3.2 Protein digestibility of wheat (Triticum aestivum) and durum wheat (Triticum durum)
An overview of wheat (T. aestivum) and durum wheat (T. durum) is presented in Table 1. The protein content tends to be slightly higher in germinated wheat. Two studies indicate that germination has a minor negative effect on IVPD, whereas one study has reported a considerable increase in germinated wheat.
Montemurro et al. (2019) reported that germination significantly improved IVPD and decreased the concentration of condensed tannin and phytic acid in durum wheat. Also, a significant increase of total free amino acids was observed with nearly tenfold increase from 760 to 7033 mg/kg. However,
the highest IVPD was achieved with sourdough fermentation alone. The decrease of phytic acid is an agreement with Singkhornar et al. (2013). They also discovered significantly higher protein content in germinated wheat, which is an agreement with Zhu et al. (2017). Similar results have been reported in other studies as well (Kavitha & Parimalavalli, 2014; Lemar & Swanson, 1976; Ranhotra, Loewe,
& Lehman, 1977).
Hung et al. (2012) studied the effect of germination time on amino acid composition of waxy wheat.
They discovered a significantly higher concentration of every amino acid during the germination.
However, the duration of germination affected the concentrations. For example, the concentration of lysine was highest after 6 and 24 h, but the concentration decreased below the initial level after 48 hours. The amount of total free amino acids increased significantly from 2207 to 7881 mg/kg after 48 h germination, which is in line with the observations from durum wheat by Montemurro et al.
(2019) and wheat by Tkachuk (1979). Tkachuk (1979) also discovered that the concentration of lysine increased throughout the germination process.
Digestion of wheat and durum wheat proteins release resistant peptides that can cause adverse effects to some individuals (Graziano et al., 2019; Mamone et al., 2015; Pilolli et al., 2019; Prandi et al., 2014; Smith et al., 2015). Germination has been studied as a method to decrease the number of these peptides after digestion (Boukid, Prandi, Buhler, & Sforza, 2017; Boukid, Prandi, Vittadini, Francia,
& Sforza, 2018). Germination of durum wheat appeared to significantly reduce the amount of both immunogenic and toxic gliadin originated peptides after in vitro digestion. However, these peptides were still present and thus consuming germinated wheat is not safe for individuals with celiac disease.
The germination time affected the amount of detected peptides and the most notable degradation was observed after 6 days (Boukid et al., 2018). This finding is in line with results from Koehler et al.
(2007) who detected that gluten of common wheat substantially degraded during 7 days of germination and total gliadins started to degrade after 4 days.
Germinated wheat has been utilized to improve the nutritional quality of different types of food products. Świeca et al. (2017) discovered a significantly higher relative protein digestibility and the amount of free amino acids and peptides in sprouted wheat flour compared to raw. However, the addition of sprouted flour did not affect the relative protein digestibility in breads, and the addition in fact significantly lowered the amount of free amino acids and peptides. Addition of sprouted flour increased the protein content significantly when 10–20% wheat flour was replaced. Nevertheless, IVPD was significantly reduced in these breads.
Addition of prolyl endopeptidase or enzymes produced by lactic acid bacteria has been reported to decrease immunoreactive peptides during bread making and in vitro digestion (Brzozowski, 2018).
In fact, sourdough fermentation with the addition of proteases can be used to produce gluten-free wheat bread (Rizzello et al., 2007). This type of bread has been reported to have significantly higher IVPD than wheat containing bread and commercial gluten free breads (Rizzello, Montemurro, &
Gobbetti, 2016). The addition of bacteria originated phytase into wheat flour has been observed to effectively increase IVPD (Tripathi, A, & Kapoor, 2018). High-phytase yeast is also a potential method to reduce phytate in wheat products (Haraldsson, Veide, Andlid, Alminger, & Sandberg, 2005). Baking on the other hand, reduces wheat gluten digestibility and solubility (Smith et al., 2015).
However, heat treatment of wheat products has been reported to lower the wheat’s allergenicity when compared to raw flour (de Gregorio et al., 2009). Replacing some of the wheat with gluten-free cereal flour is also a promising way to reduce the adverse effects of wheat products (Susanna &
Prabhasankar, 2012). Additives such as hydroxypropylmethyl cellulose, xanthan gum or locust bean gum can be added to improve pasting properties, texture, and sensory quality, even though these additives tend to lower IVPD.
Whole wheat flour can be fractioned to process branny fraction separately to acquire nutritional improvement of whole wheat products (Demir & Elgün, 2014). Stabilization of branny fraction with autoclaving, microwaving, infrared and ultraviolet C irradiation were discovered to significantly increase wheat bread’s IVPD and decrease its phytic acid content. IVPD of wheat products can also be improved by the addition of other type of cereal bran or husk (El-Moniem, 1994). Fermentation with added xylanase is also an effective way to achieve higher IVPD of different types of bran (Pontonio et al., 2020). Along with higher IVPD, supplementation of wheat bread with fermented bran significantly improve several nutritional characteristics, such as protein, fiber, ash, and total phenolic contents.
Mandalari et al. (2018) reported that particle size affects the digestibility of durum wheat porridges, the smallest particle size being the most digested. Furthermore, storing conditions can affect IVPD.
Rehman & Shah (1999) reported a decrease in lysine content and IVPD of wheat in 25 and 45℃
temperatures, whereas only minor changes were observed at 10℃. Cultivar is also an important factor in wheat IVPD. Gulati et al. (2020) reported significantly higher IVPD in modern cultivars compared to historical cultivars.
Table 1. Effects of malting/germination on protein content and in vitro protein digestibility of wheat (Triticum aestivum) and durum wheat (Triticum durum).
Reference Germination Raimondi et al., 2017 Fresh
durum
Mandalari et al., 2018 Durum wheat
35.1–62.6
Tripathi et al., 2018 Wheat 11.2 48.83
Montemurro et al., 2019 Durum wheat
3 d, 16.5°C 17.46 17.34 52.6 78.9
IVPD: In vitro protein digestibility. R: Raw; G: Germinated; NS: scientific species not specified.
Bolded numbers indicate statistically significant differences between raw and germinated wheat.
3.3 Protein digestibility of barley (Hordeum vulgare)
An overview of barley (H. vulgare) is presented in Table 2. As the table indicates, protein content varies between 11.4–14.2% and 10.4–13.61% in native and germinated barley, respectively.
Additionally, germination appears to increase protein digestibility.
Bai et al. (2018) reported that untreated hull-less barley contained 11.4 g/100 g of crude protein and that lysine was the first limiting amino acid with a concentration of 0.39 g/100 g of flour with an AAS of 0.61. The IVPD was 72.30% and the IV-PDCAAS 44.44%. Montemurro et al. (2019) discovered a statistically significant increase of IVPD with barley from 54.5 to 83.7% after germination. A slight
decrease in protein content was observed as well as a significant increase in total free amino acid content from 1475 to 8119 mg/kg. A significant reduction of phytic acid was also reported.
Singkhornart, Gu, & Ryu (2013) reported a higher protein content in barley after germination. Also, a significant decrease in phytic acid content and a significant increase in protease activity were reported as affected by germination. Chung, Nwokolo, & Sim (1989) did not discover a difference in the protein content in raw and sprouted barley, but they reported a significant increase in lysine content after sprouting. They also reported significantly higher in vivo protein digestibility with rats.
However, Beloshapka et al. (2016) reported distinctly lower lysine content in malted barley than in any other barley products.
Germinated barley has been used as an ingredient of food products and germination is often combined with other processing techniques (Arora, Jood, & Khetarpaul, 2010; Bai et al., 2018; Montemurro et al., 2019; Singkhornart et al., 2013). Gahlawat & Sehgal (1994) compared the protein digestibility of weaning mixes based on roasted or malted products. For both wheat- and barley-based weaning mixes a significantly increased IVPD was observed. Malting also improved IVPD more effectively than roasting. Arora, Jood, & Khetarpaul (2010) investigated the effects of different processing techniques on barley-based food mixtures. The germination alone resulted significantly higher protein content compared to a product that were germinated, autoclaved, and fermented. Additionally, the amount of lysine increased as an effect of germination and the highest lysine content was measured from the germinated, autoclaved, and fermented food mixture. Montemurro et al. (2019) reported the highest IVPD of barley from combination of germination and sourdough fermentation. Other techniques can be used to increase IVPD of barley, for example infrared heating and tempering (Bai et al., 2018).
Table 2. Effects of malting/germination on protein content, in vitro protein digestibility and content of some antinutritional factors of barley (Hordeum vulgare).
IVPD: In vitro protein digestibility; TIA: Trypsin inhibitor activity; TIU: Trypsin inhibitory units; R: Raw; G: Germinated; NS: scientific species not specified; DM: Dry mass.
Bolded numbers indicate statistically significant differences between raw and germinated barley. *In vivo protein digestibility.
Reference Germination
conditions
Protein content % IVPD % Lysine Condensed tannins
(mg/g of sample)
TIA (TIU/mg of sample)
Phytic acid/phytate (g/100 g of sample)
R G R G R G R G R G R G
Chung et al. 1989 Barley (NS) 6 d, 22℃ 11.85 11.85 65.28* 79.69* 0.32 (%
of protein)
0.40
Singkhornart et al., 2013
Barley (NS) 25℃ 11.89 12.43 <1.2 <1.0
Beloshapka et al., 2016
Barley 13.5 0.06
% of DM Raimondi et al.,
2017
Fresh barley sprouts
6 d, 25℃ 10.4
Bai et al., 2018 Hull-less barley (NS)
11.4 72.3 0.39 (g /
100g flour)
1.99 1.25
Montemurro et al., 2019
Barley 3 d, 16.5°C 14.02 13.61 54.5 83.7 0.26 0.24 0.82 0.78 0.73 0.36
3.4 Protein digestibility of faba bean (Vicia faba L.)
An overview of faba bean (V. faba L.) is presented in Table 3. Protein content in germinated faba bean tends to be higher than in raw. Germination seems to increase IVPD and has a clear impact on antinutritional factors.
Sulphur containing amino acids, i.e. methionine and cysteine, are the first limiting amino acids in faba bean (Alghamd, 2009; Nosworthy et al., 2018). Additionally, tryptophan has been reported to be the first limiting amino acid in some cases (Nosworthy et al., 2018; Setia et al., 2019). Setia et al.
(2019) discovered that after germination threonine was the first limiting amino acid. In the same study a significant increase in IVPD after 72 h germination was reported. However, the increase did not significantly affect IV-PDCAAS which was 56.2 % before and 56.5 % after germination. One older study reported minor decrease in the proportion of methionine, cysteine, and threonine from the total amount of amino acids after germination (Hsu, Leung, Finney, & Morad, 1980). Khalil (2001) reported a slight decrease of cysteine and an increase of tryptophan and threonine after germination.
Di Stefano et al. (2019) reported a significant increase in degree of protein hydrolysis after 5 d germination in faba bean, however, the difference was not significant after simulated gastrointestinal digestion.
Khalil (2001) observed a significant increase in true digestibility of protein in vivo with germinated faba bean. The number increased 10 %-units from 78 to 88% and was close to the casein control diet which was 92%. Protein efficiency ratio and biological value were also improved significantly after germination. Another in vivo study demonstrated a significantly improved apparent nitrogen digestibility in faba bean based diet, which increased from 53.6 to 58.3% due to germination, and net protein utilization, which increased from 61 to 75% (Rubio, Muzquiz, Burbano, Cuadrado, &
Pedrosa, 2002). Germination also improved apparent ileal digestibility of all amino acids studied.
These findings are in line with another in vivo study, where germinated faba bean product exhibited better protein efficiency ratio, true nitrogen digestibility, biological value, and net protein utilization than other two products (Bakr & Bayomy, 1997). Rubio (2003) has reported similar results with other in vivo studies.
Germination has been reported to affect the amounts of antinutritional factors in faba bean. Setia et al. (2019) observed a significant reduction in contents of condensed tannins and phytic acid, and in the activity of trypsin inhibitor. These findings are in line with other studies (Kassegn, Atsbha, &
Weldeabezgi, 2018; Khalil, 2001; A. Sharma & Sehgal, 1992). The reduction has been reported to be up to 75% for phytic acid (Khalil, 2001), 65% for TIA and from 80 to 91% for tannins (Kassegn et
al., 2018; A. Sharma & Sehgal, 1992). Vidal-Valverde et al. (1998) reported a 45% reduction of phytic acid after germination.
Fermentation is an effective method to improve the utilization of faba bean protein. Coda et al. (2017) discovered that complementing wheat bread with faba bean increased protein content. The addition of sourdough fermentation to the process improved IVPD and nutritional quality indices such as essential amino acid index (EAAI), biological value (BV), protein efficiency ratio (PER), and nutritional index (NI). The increase of IVPD and BV were statistically significant. These results are in line with discoveries by Sozer et al (2019). They obtained significantly higher IVPD, protein score, EAAI, BV and NI from bread made of sourdough pre-fermented faba bean flour compared to breads from unfermented faba bean and soybean flours. Similar results have been reported by Rizzello et al.
(2017) with pasta fortified with fermented faba bean. Verni et al. (2019) discovered that lactic acid fermentation increased IVPD and concentration of free amino acids of faba bean flour. Several bacterial strains can induce positive effects on IVPD and reduction of antinutritional factors (Rizzello et al., 2019). Rosa-Sibakov et al. (2018) reported slight, insignificant decrease of phytic acid after 24 h fermentation of faba bean. Compared to this result, phytase treatment of native faba beans proved to be a more effective method to reduce phytic acid. The phytase treatment also increased protein solubility in the lower end of the pH-scale and improved IVPD especially during the gastric phase.
Contradictory to previous results, Chandra-Hioe et al. (2016) did not discover significant differences in IVPD or TIA between native and fermented faba beans.
Another method for more effective utilization of faba bean protein is fractionation. Vogelsang-O'Dwyer et al. (2020) compared the IVPD of dry fractioned protein-rich faba bean flour, acid extracted protein isolate and dehulled flour. The isolate demonstrated significantly higher IVPD and lower TIA. However, protein solubility of the dry fractioned product was higher. A combination of air classification and fermentation has been studied by Coda et al. (2015). Fermentation significantly improved IVPD of unfractioned faba bean flour and starch rich fraction. TIA was significantly reduced in flour and protein rich fraction after germination. Amount of tannins, vicine and convicine were reduced in flour and both fractions after fermentation. Fermentation did not affect the phytic acid content, which was, however, significantly lower in starch rich fraction.
Impacts of extrusion, boiling and baking on faba bean protein quality were studied by Nosworthy et al. (2018). Baking resulted highest AAS, DIAAS and IVPDAAS values despite lower IVPD.
Sulphur-containing amino acids were limiting amino acid after all treatments, and additionally tryptophan for cooked faba bean. Cooking has been reported to significantly decrease tannin and phytic acid content of faba bean and increase IVPD (A. Osman et al., 2014). In addition to heating,
soaking in sodium carbonate has been reported to reduce tannin content and improve IVPD (Babiker
& el Tinay, 1993). Furthermore, citric acid soaking in combination with cooking and dry-heating without soaking have been observed to significantly decrease phytic acid content (Vidal-Valverde et al., 1998). Alonso et al. (2000) reported that dehulling, soaking and extrusion significantly reduced the amount of condensed tannins in faba bean. Furthermore, soaking and extrusion significantly reduced phytic acid content, whereas it was increased after dehulling. Dehulling excluded, all the treatments significantly improved IVPD, extrusion being the most effective. Dehulled faba bean in fact had higher TIA, chymotrypsin inhibitor activity, and a-amylase inhibitor activities than raw seeds whereas all other treatments lowered them.
Soaking, autoclaving, and cooking have been reported to decrease the amount of phytic acid in faba bean (Khalil, 2001). This was also demonstrated with in vivo experiment with rats, as all the treatments significantly improved PER, true digestibility (TD) and BV of the diet of rats compared to raw faba bean. A study focused on hydrothermal treatments reported that annealing significantly improved IVPD of faba bean. (Chávez-Murillo, Veyna-Torres, Cavazos-Tamez, de la Rosa-Millán,
& Serna-Saldívar, 2018). Microwave treatments have also been reported to affect IVPD (Pysz, Polaszczyk, Leszczyńska, & Piątkowska, 2012). IVPD increased and TIA decreased as the soaked faba beans were subjected to higher amounts of energy. However, increase in the energy also decreased protein solubility. Khatoon & Prakash (2004) reported 77,4 % IVPD for faba bean, which was slightly but significantly lower than pressure cooked faba beans. The study did not report the IVPD of raw product. Cultivation conditions can affect IVPD of faba beans as Babiker et al. (1995) have reported. Nitrogen fertilization and fixation caused an increase in protein content with a slight decrease in IVPD. Additionally, viral infection was reported to decrease IVPD.
Table 3. Effects of germination on protein content, in vitro protein digestibility and content of some antinutritional factors of faba bean (Vicia faba L.).
Reference Germination
IVPD: In vitro protein digestibility; TIA: Trypsin inhibitor activity; TIU: Trypsin inhibitory units; R: Raw; G: Germinated.
Bolded numbers indicate statistically significant differences between raw and germinated faba bean. *In vivo digestibility. **Fresh weight. ***Scientific species not specified.
3.5 Protein digestibility of other cereals and legumes Cereals and pseudocereals
An overview of the effects of germination on cereals and pseudocereals is presented in Tables 4-6.
In many studies, germination conditions affect the protein content and/or IVPD. The values presented in Tables 4-6 are either ranges of germination’s outcome or exhibit the largest difference reported.
As Table 4 indicates, all the studies with sorghum (Sorghum bicolor) have reported a decrease in protein content after germination. However, a clear tendency of higher IVPD after germination can also be observed. Furthermore, decreased amounts of antinutritional factors have been reported (Elkhalil, El Tinay, Mohamed, & Elsheikh, 2001; Elmaki, Babiker, & El Tinay, 1999; Mohamed Nour, Mohamed Ahmed, Babiker, & Yagoub, 2010).
Table 5 indicates that germination has a clear positive effect on millets, as the most studies included reported higher protein content and IVPD compared to native grains. Results from the few studies with rice (Oryza sativa) are inconclusive presented in Table 6. Albarraccín et al. (2019) reported a 30% decrease in phytic acid in addition to a clear improvement of IVPD. The reduction of phytic acid is an agreement with Cornejo et al. (2015), even though they observed only slight differences in IVPD of rice breads. As with rice, only a few studies about maize (Zea mays), amaranth (Amaranthus spp.), quinoa (Chenopodium quinoa), and hull-less oats (Avena nuda) were included in this review and the results are therefore inconclusive. Nonetheless, a reduction of antinutritional factors has been reported in all these species (Fageer, Babiker, & El Tinay, 2004; Gernah, Ariahu, & Ingbian, 2011; Hejazi, Orsat, Azabi, & Kubow, 2016; Montemurro et al., 2019; Tian et al., 2010).
Table 4. Effects of malting/germination on protein content and in vitro protein digestibility of sorghum (Sorghum bicolor).
Reference Product Protein content % IVPD %
Other findings
R G R G
Malleshi & Klopfenstein, 1998
Sorghum 11.8 10.6–11.7 Protein content of rootlets
decreased during germination.
Elmaki et al., 1999 Sorghum (NS)
Longer soaking and germination times both decreased IVPD for
Longer soaking and germination times both decreased IVPD for