A Short Review: Bioactivity of Fermented Rice Bran

: Rice bran is a by-product of the rice milling process, which refers to the processing of brown rice into polished rice. It contains a considerable amount of functional bioactive compounds. However, the utilization of these compounds is limited and calls for an effort to ferment rice bran. One of the methods that can significantly increase the added value of rice bran as well as its bioactivity is the solid-state fermentation. It can also be one of the strategies that help in the production of rice bran as a functional ingredient with higher bioactivity for health promotion.

the added value of rice bran for further use in product development fermentation, especially solid-state fermentation SSF . SSF is a fermentation technique that can escalate the bioactive compounds in food, such as the phenolic content, thereby contributing to antioxidant activity 13 15 . Mold, yeast and bacteria are the types of microorganisms that are often used in the SSF method. Mold species are suitable for use in SSF because they are capable of producing enzymes like amylase, pectinase, xylanase, cellulase, chitinase, protease, lipase and β-galactosidase 14 . Therefore, SSF helps to increase the bioactive compounds of plant materials. In addition, fermentation using the SSF method can improve the sensory profile of the rice bran 13 .
SSF is currently used in a wide range of applications from traditional applications such as tempe fermentation in Indonesia. Recently, it has been used to develop new food ingredients like bioactive compounds. It was also a new trend regarding bioethanol and biodiesel production as new energy sources that used agricultural by-products as substrate with microbe as starter. SSF has the following advantages: 1 It is a promising method for obtaining a high yield of bioactive compounds that require less water for microorganisms growth; 2 It can convert cheap agroindustrial by-products into products that contain various valuable compounds; 3 It can minimise microbe contaminants; 4 It is a simple processing technique and is more practical; 5 It involves high productivity; and 6 Its media conditions resemble the actual habitat 14 .
This review discusses the bioactivity of fermented rice bran, which has the potential to be developed as a food ingredient. The discussion begins by defining fermented rice bran, the active compounds contained therein, both volatile and non-volatile compounds and in-vivo examination to show fermented rice bran s bioactivities.

Fermentation can increase the active compounds of rice bran
Fermentation has been widely used to improve the quality of food. It is popular thanks to the extended storage period that it offers, which improves taste and increases the bioactive compounds and protein content of the food item 11,16,17 . Several studies have shown that fermented rice bran has a higher content of bioactive compounds and better functional properties than non-fermented rice bran. The increase in phenolic compounds after the fermentation process occurs due to the breakdown of complex compounds that bind to lignocellulose or polysaccharides 18,19 . During the fermentation process, the microbes as starters produce enzymes that can hydrolyse complex compounds in the bound form into compounds in the free form 19 . Besides being able to increase the bioactive compounds and antioxidant activity, the fermentation process can improve the sensory profile of rice bran. Cempo Ireng and  Inpari 30 fermented rice bran with Rhizopus oligosporus  for 72 hours are more acceptable by the 75 naïve panellists  than the benchmarks non-fermented rice bran because  the sample had a dominant liking of both aroma and  taste 20 .
Compared to non-fermented rice bran, rice bran fermented with Rhizopus oryzae for 24 hours can increase the total phenolic compounds TPC up to five times and inhibit 2,2-diphenyl-1-picrylhydrazyl DPPH free radicals up to 87 21 . In addition, the TPC in fermented rice bran using Rhizopus oryzae for 120 hours increased from 2.4 mg/g to 5.1 mg/g 13 .
The fermentation process aims to increase the nutritional content and bioactive compounds in rice bran. Several studies have been conducted to determine the effects of fermentation on the nutritional content, antioxidant activity and bioactive compounds in rice bran. The fermentation process is carried out using molds, such as Monascus pilosus 11 29

and mushrooms
Pleurotus sapidus 30 . SSF is a fermentation method effective for increasing antioxidant activity and TPC in food with solid media 31 . The mold Rhizopus sp. and Aspergillus sp. are proper cultures that can be used in the SSF method since they do not produce toxic compounds during fermentation 32 . Some of the research results regarding fermented rice bran are shown in Table 1. Some of the fermentation processes of rice bran were undertaken using cultures like Rhizopus oryzae, Rhizopus oligosporus, Lactobacillus plantarum, Monascus pilosus, Aspergillus kawachii, lactic acid bacteria and Saccharomyces boulardii.
Using lactic acid bacteria starter and Rhizopus oryzae in fermented rice bran produces lactic acid 33 . However, the use of Rhizopus oryzae is more desirable owing to the easy separation between the mold biomass and the substrate. Several studies have reported that the fermentation process in rice bran can increase the bioactive compounds. This occurs due to the production of extracellular enzymes which affect the increase in the bioactive compounds in the substrate 17 . Furthermore, the microorganisms used as starters in the fermentation process will synthesise compounds and activate metabolic pathways to adapt to the substrate 34 .
Fermented rice bran using Rhizopus oryzae increases the ash, fibre, protein, amino acids, phospholipids and the total phenolic content. The increase in ash in fermented rice bran is due to the synthesis of mycelia 35 . Another study showed that the increase in the fibre content is due to the intrinsic production of chitin 17 which is one of the compounds of the hyphal cell wall on Rhizopus oryzae 36 .
Rhizopus oryzae also produces phytase that hydrolyses complex proteins leading to an increase in dissolved protein 36 . Phytic acid degradation by Rhizopus oligosporus can increase the content of minerals, such as iron, magnesium and zinc, which are bound to phytates 37 . The presence of phytase also accelerates the hydrolysis of phytic acid, thereby affecting the decrease in its content 17 . This further leads to the enhancement of phenolic compounds because of the degradation of lignocellulose by the enzymes in Rhizopus oligosporus 20 . The increase in phenolic compounds also occurs in fermented rice bran with the use of Rhizopus oligosporus and Aspergillus kawachii combined with lactic acid bacteria Lactobacillus brevis, Lactobacillus rhamnosus, Enterococcus faecium 28,38 . Several studies have also shown a decrease in moisture, lipid and phytic acid levels. An increase in the temperature during fermentation causes a decrease in water content 17 . The lipids are used by fungi to form mycelia, resulting in decreased lipid levels 36 .
Rhizopus oligosporus is the main mold and is widely used as a starter in making tempe in Indonesia. Studies on fermenting rice bran with Rhizopus oligosporus have been reported by several researchers 23,28 . The research revealed a similar increase in phenolic compounds in fermented rice bran, which occurs due to the activity of hydrolytic enzymes of fungi such as β-glucosidase 25 ; these enzymes can increase the hydroxyl molecule, which can result in a rise in the amount of free phenolic compounds  38) in the rice bran 39 . The use of mixed cultures Rhizopus oligosporus, Rhizopus oryzae and other mixtures in the fermenting of rice bran has also been reported. Fermenting rice bran using the Rhizopus oligosporus starter produced the highest DPPH radical scavenging activity, with a fermentation time of 96 hours on Inpari 30 cultivar 20 .

Volatile compounds of rice bran
Volatile compounds play a role in the formation of aroma in food products. They give either a pleasant or unpleasant aroma. They can form along the food chain, starting after harvesting, and occur throughout post-harvest handling, distribution and storage. The heating treatment can result in the loss of volatile compounds due to their volatility. Several previous studies have discussed the volatile compounds found in rice and rice bran. The compounds that have been identified include alcohol, alkanes, ketones and aldehydes as shown in Table 2.
Our group has identified and compared the volatile compounds of fermented and unfermented Inpari 30 and Cempo Ireng rice bran 20 . The extraction of volatile compounds was carried out using the headspace solid-phase microextraction method and analysed using the Gas Chromatography-Mass Spectroscopy GC-MS instrument. The analysis showed that there were 57 volatile compounds, including alcohol 23 , aldehyde 19 , acid 11 , ketones and esters 9 , ester 7 , terpenes and benzene 5 , furans and lactones 3 and pyridine, as well as pyrazine and thiazole 2 . The formation of volatile compounds happens as a result of the lipid degradation reaction during fermentation and the Maillard reaction due to the sterilisation process. Most of the volatile compounds in rice bran under fermentation treatment originate from the lipid degradation reactions. In Inpari 30 cultivar, they were 3-methyl-3-butenol, 2,3-butandienol, benzylalcohol, glycerine, methyl hexadecanoate, E -9-methyl octadecanoate, Z,Z -9,12-methyl octadecadienoate, 1R-α-pinene, caryophyllene, 2-methoxyphenol and 3-methyl pyridine. However, in fermented Cempo Ireng rice bran cultivar, the volatile compounds were dominated by 4-methyl-3-pentenol, benzylalcohol, glycerine, methyl hexadecanoate, ethylbenzene and caryophyllene.
The fermentation resulted in the rice bran having sweet, creamy, fatty, smoky and green aromas. Smoky and green aromas are thought to trigger the rancid aroma in rice bran due to the lipid degradation by enzymes during the fermentation process. In non-fermented rice bran, the volatile compounds are mostly formed by the Maillard reaction which is influenced by the sterilisation process. In the fermented Inpari 30 rice bran cultivar, the volatile compounds that are thought to be dominant include 2-furanmethanol, nonanal, methyl tetradecanoate, phenol and 2-methoxy-4-vinylphenol. Meanwhile, 2-furanmethanol, hexanal, naphthalene, 1R-α-pinene and 2-methoxy-4-vinylphenol are the volatile compounds in fermented Cempo Ireng rice bran. These compounds are thought to form a burnt, nutty and fatty smell. In the non-fermented rice bran, hexanal and nonanal compounds were also found, obtained from the lipid degradation reaction which is thought to produce a rancid aroma. Apart from that, other research 43 state that 4-vinylphenol is the main component that causes the unpleasant odour. The difference in the volatile compounds of several rice varieties, such as Inpari 30 and Cempo Ireng, emerges from the differences in the varieties, planting locations, nutritional contents and bioactivities in the rice bran.  References: [40][41][42] Almost the same components aldehydes, alcohols, alkenes and ketones were also found in red rice and black rice bran cultivars Table 3 . The β-ocimene component in red rice bran is known to have an aroma like damp clothes 44 . Meanwhile, myristic acid compounds are known to carry a waxy and fatty aroma 45 , and the compounds 6,10,14 trimethyl-2-pentadecane are known to have an aroma of oil, celery and wood 46 .
The guaiacol component is reported to be the main cause of the aroma of black rice. In black rice bran, the nonanal compound is known to have a citrusy, green and fatty aroma 47 . Meanwhile, caproic acid and pelargonic acid have a fatty, cheesy and waxy aroma 48,49 .

Non-volatile compounds of rice bran
Non-volatile compounds of rice bran have been report-ed 50 on the Calrose, Dixiebelle and Neptune rice varieties that grow in the south-eastern part of California, United States. In this study, 465 metabolites were found, consisting of amino acids 126 , carbohydrates 35 , vitamins and cofactors 28 , energy 11 , lipids 137 , nucleotides 40 , peptides 28 , secondary metabolites 55 and xenobiotics 8 . The antioxidant compounds of rice bran include amino acids 4-guanidinobutanoate and taurine , vitamins and cofactors tocopherols and tocotrienols and secondary metabolites ergothioneine and quinates 50 which can potentially be used as antioxidants to inhibit chronic diseases and infections. Other studies have shown an increase in metabolite diversification in fermented rice bran when using Saccharomyces boulardii. The use of specific rice varieties and fermentation treatment on rice bran affects the amount and type of active compounds produced 5,17,51 . Several studies related to the identification of non-volatile compounds in rice bran are presented in Table 4. Table 3 Volatile compounds in red and black rice bran cultivars.

Rice cultivars
Volatile compounds
Non-volatile compounds in fermented rice bran using Rhizopus oligosporus in Inpari 30 and Cempo Ireng rice bran varieties have also been reported 57 . Fermentation was carried out for 72 hours at room temperature of 30 with SSF. The analysis of non-volatile compounds was carried out using the ultra-performance liquid chromatography-tandem mass spectrometer UPLC-MS/MS and electrospray ionisation ESI mass spectrometry in positive ion mode. There were 72 compounds identified and categorised into secondary metabolites 50 , lipids 22 , amino acids 11 , vitamins and cofactors 10 , peptides 4 , nucleotides 1 and carbohydrates 1 . Fermentation in Inpari 30 and Cempo Ireng rice bran produces new compounds from the metabolism of tyrosine, phenylalanine, pentatonic acid, dipeptides and sphingolipids and terpenoids. The analysis also showed that adenosine was the most dominant non-volatile compound in the two types of rice bran cultivars.

In-vivo study of fermented rice bran
The parameters that are widely tested in the in vivo study of fermented rice bran are total cholesterol TC , triglycerides TG , aspartate aminotransferase AST , alanine aminotransferase ALT , serum blood sugar levels and blood pressure. Fermented rice bran is a good source of antioxidants owing to its bioactive compounds such as phenolic compounds, flavonoids, carotenoids and anthocyanins. Many of these bioactive compounds are known to be useful as functional ingredients to prevent various chronic diseases.
The studies in vivo on fermented rice bran are presented in Table 5. The hepatoprotective effect on the administration of 0.4 w/w fermented rice bran orally in induced mice carbon tetrachloride CCl 4 58 . The results of these studies indicate that fermented rice bran can prevent the incidence of liver damage caused by CCl 4 by increasing the antioxidant activity and decreasing AST and ALT. Another study showed the same results, i.e., a decrease in AST and ALT in the group of mice given fermented rice bran 59 .
AST has been widely used as a marker to measure liver damage 58 . TNF-α, IL-6 and IL-1β are pro-inflammatory cytokines that can cause colonic tissue damage and ulceration of the large intestine if present in excessive amounts 60 . The supplementation of fermented rice bran can protect the C57BL/6N mice from ulcerative colitis by decreasing TNF-α, IL-6 and IL-1β, increasing the body weight and stool consistency and reducing intestinal bleeding due to a rise in short-chain fatty acids SCFA and tryptamine 59 . Another benefit of fermented rice bran is its anti-inflammatory effect which is achieved by reducing pro-inflammatory cytokines. The ovalbumin OVA model showed that fermented rice bran extract reduced TNF-α, interferon IFN-γ , IL-6 and IL-10 in mice 61 . Another study states that fermented rice bran extract can prevent atopic dermatitis by reducing T cells CD8 and cells Gr-1 /CD11b B and inhibiting the expression of cytokine mRNA IL-5 and IL-13 62 .
Fermented rice bran has been reported to lower blood pressure when administered to stroke-prone spontaneously hypertensive rats SHRSP 28,38,57 . Both studies reported that the oral administration of fermented rice bran can reduce systolic blood pressure sBP , improve blood sugar levels, reduce insulin resistance and increase nitric oxide NO in the blood 28,57 . Furthermore, fermented rice bran can effectively lower blood pressure due to an increase in the inhibitory activity of the angiotensin-converting enzyme ACE in serum 38 .
TC, TG, LDL cholesterol and high-density lipoprotein HDL cholesterol are an important part of the lipid fraction in the human body. The anti-hypercholesterolemic effect of fermented rice bran has also been reported 63 . Rice bran compounds such as γ-oryzanol can lower the cholesterol levels in the blood, lower the LDL cholesterol, and increase the HDL cholesterol. Mice orally administrated with fermented rice bran showed a decrease in TC, TG and LDL and increased blood HDL cholesterol 38,59,63 . Several possible mechanisms can occur in the improvement of lipid fractions in the blood, such as a decrease in lipid and cholesterol absorption as well as cholesterol synthesis and an increase in cholesterol secretion, HDL synthesis or antioxidant activity 63 .

Conclusions and perspectives
Fermentation using the SSF technique is an alternative method that can be used to increase the number of active compounds in rice bran as a by-product of the rice milling process. The biochemical reactions that occur during the fermentation process lead to changes in the nature and characteristics of the rice bran. SSF can increase the TPC of rice bran. The volatile and non-volatile compounds present in the rice bran contribute to its aroma and flavour, which are formed during the fermentation process, and can improve the sensory quality of the rice bran. The increase in the number of active compounds in the rice bran is accompanied by a rise in bioactivity, which has been proven in the in vivo studies.
SSF is a simple technique for producing bioactive compounds in rice bran. This technique is economical because the materials used are agro-industrial by-products. Additionally, this technique is environment-friendly since it reduces the number of industrial by-products disposed into the environment. SSF can be used as an alternative method to improve the functional properties of rice bran as a food ingredient for health promotion in the future.  Note: ↑ = increasing; ↓ = decreasing