COMPOSITION OF A PRESERVATIVE FREE FERMENTED FLOUR AND METHOD OF PREPARING THE SAME THEREOF

The present invention generally relates to the food industry. Most precisely, the present invention relates to a preservative free fermented flour and a method of preparing the preservative free fermented flour with extended shelf life thereof.

In food sectors major task to be addressed is the nature of the product whether synthetic, chemical or natural. Many chemical and synthetic preservatives are taking the major place in the market. These products have enormous side effects along with reducing final food product’s taste, texture and quality. As health insight is increased in the world for a healthy lifestyle, people are looking for natural, clean-label, and safe alternatives.

The foremost market for food preservatives is flourishing widely in baking industry as the bakery products have a very brief shelf life and the quality of the products depends on the time interval between baking and consuming the products. The main spoilage of bakery products takes place largely due to the growth of moulds, specifically with the genera Aspergillus, Fusarium and Penicillium, and also, because of the roping of bakery products, caused by Bacillus sp., especially B. subtilis and B. licheniformis. Hence the use of preservatives ha exponentially increased in past few years without realizing and considering the side effects caused by these additives and stabilizers.

Out of all, the most commonly used preservative in baking industry is calcium propionate which is obtained by the chemical synthesis propionic acid and calcium carbonate or calcium hydroxide as the main raw materials. However, it has been reported that addition of calcium propionate in a bakery product may cause adverse effects, such as headaches and migraines (Malinee Pongsavee, 2019).

Another human study linked propionate intake to the increased production of insulin and glucagon, a hormone that stimulates glucose (sugar) release. This can lead to insulin resistance, a condition in which the human body cannot use insulin properly, and may lead to type 2 diabetes (Tirosh et al; 2019).

In addition, a study in 27 children found that some experienced irritability, restlessness, poor attention, and sleep issues after consuming calcium-propionate-containing bread daily (S Dengate and A Ruben; 2002).

Hence, in order to avoid the preservatives and develop raw materials devoid of any chemical preservative is a dire need of the time. One way of avoiding the use of preservative is the use of fermented flour which are prepared using specific bacterium and yeast and do not contain any additives, preservatives and stabilizers. In other words, fermented flour is a mixture of flour (from wheat, rye, rice, etc.) and water, which is fermented by the action of various bacteria and yeasts. The fermented flour provides baked products with extended shelf life and meets consumer’s demand for natural food without additives. However, it’s a challenge to prepare a preservative free fermented flour with extended shelf life because of the lack of adequate knowledge of preparing the fermented flour without adding any preservative and additives.

Hence, there is a dire need to prepare a fermented flour without preservatives to be used in bread making that would extend the shelf life of bread without preservatives without any adverse alterations of bread quality.

The present application aims to provide one or more new and useful methods for making and/or using fermented or sour flour. Essential features of the application are provided by one or more independent claims, whilst advantageous features are presented by their dependent claims respectively.

It is therefore an object of the present embodiments to provide a preservative free fermented or sour flour, which is free from calcium propionate and can be used for baking directly to produce sour dough products without any bacterial fermentation by the user. It is further an object of the present embodiments to provide a method for producing the preservative free fermented flour which is easy to use and economical.

Another object of the present embodiments is to provide a method of preparing a preservative free food product and a kit thereof, with all the important components and instruction manual.

In order to solve the foregoing problem, an aspect of the present embodiments is to provide a composition for a preservative free fermented flour and a method of preparing the same. The composition is devoid of chemically synthesized food preservatives like calcium or sodium propionate.

In an embodiment, the present invention discloses a composition for a preservative-free fermented flour comprising all-purpose flour, salts, yeast, bread improver, a microorganism and water, wherein said plain flour is in the range of 45%-75%, more preferably 55% - 65%, salt is in the range of0.5%-2.0%, preferably 0.9%-1.5%, yeast is in the range of 0.5%-2.5%, preferably 1.0%-2.0%, bread improver is in the range of 0.30%-0.70%, preferably, 0.50%- 0.60% and water is in the range of 30%-40%, preferably, 33 %-36%.

In another embodiment, said microorganism belongs to Propionibacterium spp, preferably bacillus Acidi propioinni. The spp produces propionic acid, vitamin B 12, and other metabolites important for the baking industry.

In still another embodiment, said all-purpose flour is selected from white flour, brown flour, rye flour, spelt flour and a combination thereof. Accordingly, any kind of flour is used as per the taste and requirement of the user.

In yet another embodiment, said salts are selected from table salt, sea salt and kosher salt and a combination thereof. Salt not only perform as a flavor enhancer, but also affects the tenderness of a baked goods, which, in turn, makes the dough or batter tighter and more elastic.

In another embodiment, said yeast is selected from Saccharomyces cerevisiae. In another embodiment, pH of the flour ranges between, 2.5 to 5.0, preferably 3.2 to 4.1. In baking, pH control is critical for inhibiting spoilage by different mold species, optimum rise in yeast- and chemically leavened products and overall baked goods texture.

In still another embodiment, the proofing time for said composition is 30 minutes to 45 minutes. It is a well-known fact that under- or over-proofed bread will not rise properly when baked. In other words, if yeasted dough is not allowed to proof, the yeast cannot release carbon dioxide, and the gluten will not stretch to hold the air bubbles.

In yet another embodiment, the shelf life of the preservative free fermented flour is 15-20 days. Higher the shelf life, lower will be the degradation and mould infection of the fermented flour. In another embodiment, the present invention discloses a method of preparing the composition, comprising the steps of: i. obtaining sieved refined flour in an amount ranging from 500 grams- 1000 grams; ii. adding yeast in a ratio range of 1.5%-3.0% and flour fermented with bacillus Acidi propioinni in a ratio range of3-5%; iii. adding improver in a ratio range of 0.5%- 1.0%; iv. adding diluted fermented flour in a ratio range of 1.5%-3.0%, v. mixing the components at slow and high speed alternatively, and; vi. obtaining the preservative free fermented flour. wherein said method involves two cycles and wherein, cycle one comprises of 3%, 4% and 5% fermented flour and cycle two comprises of 1.5%, 0.75%, 3% of fermented flour and 0.75%, 1.25% and 0.375% of vinegar powder; and wherein said method has a Negative control (N) without preservatives and a positive control with calcium propionate.

In another embodiment, the weight ratio of flour to water is between 2:3 to 4:1 part and the mixture is spray dried. In still another embodiment, the pH of the fermentation mixture ranges between 3.2 and 4.1.

In another embodiment, the present invention discloses a method of preparing a food product comprising the composition, comprising the steps of: i. mixing the fermented flour as claimed in claim 1 with ice water in an amount of 290 ml 580 ml, wherein, wet mass -incorporation is mixed for 3 minutes at 24-26 ±1°C during mixing at low and high speed and continued mixing until the dough is smooth and extensible; ii. leaving the dough at the temperature ranging from 220°C- 260°C for a time ranging from 8 minutes to 12 minutes; iii. dividing the dough in small pieces and resting the dough for 5 minutes; iv. moulding and proving the dough for 50-70 min at 280°C- 320C at relative humidity 75% - 85%; v. baking the dough in a shape required at 240°C for 42-45 min; vi. cooling the dough at ambient temperature for 1-1.5 hours; and vii. obtaining and packing the food product in the form of loaves of required size.

In yet another embodiment, the present invention discloses a kit comprising the fermented flour as claimed in claim 1 and an instruction manual. The kit will comprise all the ingredients at one place and less cumbersome for the user.

The present invention is illustrated by way of example and not by way of limitation in the figures (e.g. FIG., Fig., Figure, FIG, Fig or their equivalencies) of the accompanying drawings, in which the like reference numerals indicate like elements and in which:

FIG. 1 displays a chart of bread sample distribution according to an example embodiment of the present disclosure.

FIG. 2 shows the graph of proofing time for variations to reach approximately 9.5 cm in height in cycle 1, according to an example embodiment of the present disclosure.

FIG. 3 illustrates a graph of height increased for variations during proofing in cycle 1, according to an example embodiment of the present disclosure; and FIG. 4 displays a graph of estimated time to attain 80% height increment for variations in cycle 1, according to an example embodiment of the present disclosure.

FIG. 5 shows a graph of volume, weight and specific volume for variations in cycle 1, according to an example embodiment of the present disclosure.

FIG. 6 displays a graph of bread slices left against time for visual inspection of mould growth in cycle 1, according to an example embodiment of the present disclosure.

FIG. 7 displays a graph of pH for variations throughout the 15-day shelf-life study in cycle 1, according to an example embodiment of the present disclosure.

FIG. 8 displays a graph of water activity for variations throughout the 15-day shelf-life study in cycle 1, according to an example embodiment of the present disclosure.

FIG. 9 exhibits the graphs of moisture content (%) for variations of Day (a) and throughout the 15-day study (b) Different lower-case letters indicate significant differences at P<0.05 in cycle 1, according to an example embodiment of the present disclosure.

FIG. 10 demonstrates a graph of moisture loss (%) for variations throughout the 15-day shelflife study in cycle 1, according to an example embodiment of the present disclosure.

FIG. 11 displays a graph of L* (a), a* (b) and b* (c) value for variations throughout the 15-day shelf-life study, wherein L* indicate lightness; a* indicates red/green value: positive value represents a shift towards red and b* indicates blue/yellow value: positive value represents a shift towards yellow in cycle 1, according to an example embodiment of the present disclosure. FIG. 12 shows a graphs of hardness level for variations of Day 1 (a) and throughout the 15- day study (b) Different lower-case letters indicate significant differences at P<0.05 in cycle 1, according to an example embodiment of the present disclosure.

FIG. 13 displays graphs of springiness (a) and chewiness (b) level for variations throughout the 15-day study (c) graphs of springiness (a) and chewiness (b) level for variations throughout the 15-day study. Different lower-case letters indicate significant differences at P in cycle 1, according to an example embodiment of the present disclosure.

FIG. 14 shows a graph of proofing time for variations to reach approximately 9.5cm in height in cycle 2, according to an example embodiment of the present disclosure.

FIG. 15 demonstrates a graph of height increased for variations during proofing in cycle 2, according to an example embodiment of the present disclosure. FIG. 16 shows a graph of estimated time to attain 80% height increment for variations in cycle 2, according to an example embodiment of the present disclosure.

FIG. 17 displays a graph of volume, weight and specific volume for variations in cycle 2, according to an example embodiment of the present disclosure.

FIG. 18 shows a graph of bread slices left against time for visual inspection of mould growth in cycle 2, according to an example embodiment of the present disclosure.

FIG. 19 demonstrates a graph of pH for variations throughout the 15-day shelf-life study in cycle 2, according to an example embodiment of the present disclosure.

FIG. 20 displays a graph of water activity for variations throughout the 15-day shelf-life study in cycle 2, according to an example embodiment of the present disclosure.

FIG. 20 displays a graph of water activity for variations throughout the 15-day shelf-life study in cycle 2, according to an example embodiment of the present disclosure.

FIG. 21 displays graphs of moisture content (%) for variations of Day 1 (a) and throughout the 15-day (b) study wherein different lower-case letters indicate significant differences at P in cycle 2, according to an example embodiment of the present disclosure.

FIG. 22 displays a graph of moisture loss (%) for variations throughout the 15-day shelf-life study, wherein different lower-case letters indicate significant differences at P<0.05 in cycle 2, according to an example embodiment of the present disclosure.

FIG. 23 displays graph of L* (a), a* (b) and b* (c) value for variations throughout the 15-day study wherein L* indicate lightness, a* indicates red/green value: positive value represents a shift towards red and b* indicates blue/yellow value: positive value represents a shift towards yellow in cycle 2, according to an example embodiment of the present disclosure.

FIG. 24 displays a graph Graphs of hardness level (a) for variations of Day 1 and throughout the 15-day (b) study Different lower-case letters indicate significant differences at P<0.05, in cycle 2, according to an example embodiment of the present disclosure.

FIG. 25 displays a graph of springiness (a) and chewiness (b) level for variations throughout the 15-day study (c) Graphs of springiness (a) and chewiness (b) level for variations throughout the 15-day study. Different lower-case letters indicate significant differences at P in cycle 2, according to an example embodiment of the present disclosure.

FIG. 26 displays a Radar graph of sensory attributes for variations selected for sensory evaluation, wherein mean of 1 refers to like extremely and 4 refers to neither like nor dislike and different lower-case letters indicate significant differences at P<0.05 in cycle 2, according to an example embodiment of the present disclosure.

FIG. 27 displays respondent’s opinion on ideal shelflife of bread made of natural preservatives in cycle 2, according to an example embodiment of the present disclosure.

FIG. 28 displays Respondent’s willingness to spend more cost on bread made of natural preservatives (If normal selling price at $2), according to an example embodiment of the present disclosure.

FIG. 29 displays Aspect of concerns that respondents came to mind when purchasing bread. Different lower-case letters indicate significant differences at P<0.05, according to an example embodiment of the present disclosure.

FIG. 30 displays general work process flow on day of baking, according to an example embodiment of the present disclosure.

FIG. 31 displays TPA settings and typical plot of TPA, according to an example embodiment of the present disclosure.

FIG. 32-Fig. 36 displays physical appearance of loaf produced in Cycle 1, according to an example embodiment of the present disclosure.

FIG. 37-41 displays Physical appearance of loaf produced in Cycle 2, according to an example embodiment of the present disclosure.

FIG. 42 displays Physical appearance of bread produced for sensory evaluation, according to an example embodiment of the present disclosure.

FIG. 43 displays Bread slicing, packaging, cutting and preparation for sensory evaluation, according to an example embodiment of the present disclosure.

In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances, systems and methods are shown in block diagram form only in order to avoid obscuring the present invention. Some embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, various embodiments of the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this invention will satisfy applicable legal requirements.

The embodiments are described herein for illustrative purposes and are subject to many variations. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient but are intended to cover the application or implementation without departing from the spirit or the scope of the present invention. Further, it is to be understood that the phraseology and terminology employed herein are for the purpose of the description and should not be regarded as limiting. Any heading utilized within this description is for convenience only and has no legal or limiting effect.

As used in this specification and claims, the terms “for example” “for instance” and “such as”, and the verbs “comprising,” “having,” “including” and their other verb forms, when used in conjunction with a listing of one or more components or other items, are each to be construed as open ended, meaning that that the listing is not to be considered as excluding other, additional components or items. Other terms are to be construed using their broadest reasonable meaning unless they are used in a context that requires a different interpretation.

Embodiments of the relevant invention disclose a composition of preservative free fermented flour producing several organic acids and other natural compounds in the presence of a bacterium. These organic acids can replace chemically synthesized food preservatives like calcium or sodium propionate. It is a new product the field of bakery and other food products with 100% natural ingredients.

In order to solve the foregoing problem, an aspect of the present invention is to provide a composition for a preservative free fermented flour and a method of preparing the same. The composition is devoid of chemically synthesized food preservatives like calcium or sodium propionate. The present invention clearly demonstrates that the preservative fermented flour disclosed in the present invention does not use calcium propionate and then also results into an enhanced bakery product in terms of shelf life and proofing time. Hence, the fermented flour can be used to replace the chemical preservatives such as calcium propionate.

In an embodiment, the present invention discloses a composition for a preservative-free fermented flour comprising all-purpose flour, salts, yeast, bread improver, a microorganism and water, wherein said plain flour is in the range 45%-75%, more preferably 55% - 65%, salt is in the range of0.5%-2.0%, preferably 0.9%-1.5%, yeast is in the range of 0.5%-2.5%, preferably 1.0%-2.0%, bread improver is in the range of 0.30%-0.70%, preferably, 0.50%- 0.60% and water is in the range of 30%-40%, preferably, 33 %-36%.

In another embodiment, said microorganism is a bacterium, preferably bacillus Acidi propioinni.

In still another embodiment, said all-purpose flour is selected from white flour, brown flour, rye flour, spelt flour and a combination thereof.

In yet another embodiment, said salts are selected from table salt, sea salt and kosher salt and a combination thereof.

In another embodiment, said yeast is selected from Saccharomyces cerevisiae.

In another embodiment, pH of the flour ranges between 3.2 to 4.1.

In still another embodiment, the proofing time for said composition is 30 minutes to 45 minutes.

In yet another embodiment, the shelf life of the preservative free fermented flour is 15-20 days. In another embodiment, the present invention discloses a method of preparing the composition, comprising the steps of: i. obtaining sieved refined flour in an amount ranging from 500 grams- 1000 grams; ii. adding yeast in a ratio range of 1.5%-3.0% and flour fermented with bacillus Acidi propioinni in a ratio range of 3-5%; iii. adding improver in a ratio range of 0.5%-1.0%; iv. adding diluted fermented flour in a ratio range of 1.5%-3.0%, v. mixing the components at slow and high speed alternatively, and; vi. obtaining the preservative free fermented flour. wherein said method involves two cycles and wherein, cycle one comprises of 3%, 4% and 5% fermented flour and cycle two comprises of 1.5%, 0.75%, 3% of fermented flour and 0.75%, 1.25% and 0.375% of vinegar powder; and wherein said method has a Negative control (N) without preservatives and a positive control with calcium propionate.

In another embodiment, the weight ratio of flour to water is between 2:3 to 4:1 part and the mixture is spray dried.

In still another embodiment, the pH of the fermentation mixture ranges between 3.2 and 4.1.

In another embodiment, the present invention discloses a method of preparing a food product comprising the composition, comprising the steps of: i. mixing the fermented flour as claimed in claim 1 with ice water in an amount of 290 ml to 580 ml, wherein, wet mass-incorporation is mixed for 2-5 minutes at 22-27 ±1°C during mixing at low and high speed and continued mixing until the dough is smooth and extensible; ii. leaving the dough at the temperature ranging from 220°C- 260°C for a time ranging from 8 minutes to 12 minutes; iii. dividing the dough in small pieces and resting the dough for 5 minutes; iv. moulding and proving the dough for 50-70 min at 280°C- 320°C at relative humidity 75% - 85%; v. baking the dough in a shape required at 240 °C for 42-45 min; vi. cooling the dough at ambient temperature for 1-1.5 hours; and vii. obtaining and packing the food product in the form of loaves of required size.

In yet another embodiment, the present invention discloses a kit comprising the fermented flour as claimed in claim 1 and an instruction manual.

Many modifications and other embodiments of the inventions set forth herein will come to mind of one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Moreover, although the foregoing descriptions and the associated drawings describe example embodiments in the context of certain example combinations of elements and/or functions, it should be appreciated that different combinations of elements and/or functions may be provided by alternative embodiments without departing from the scope of the appended claims. In this regard, for example, different combinations of elements and/or functions than those explicitly described above are also contemplated as may be set forth in some of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Some examples or embodiments of the relevant inventions are given below.

Materials

The materials used in bread making were: Plain flour, salt, yeast, Domino Verde - Millbo bread improver, water, preservatives and a microorganism, acidi propioni (calcium propionate/fermented flour/vinegar powder). There were 2 cycles of bread production for the shelf-life study. In both cycles, five variations were prepared whereas four variations were produced for sensory evaluation: 1.1: Cycle 1

Negative control (N) with no added preservative

Positive control (P) with calcium propionate

Test Groups A, B and C with 3%, 4% and 5% fermented flour respectively.

1.2: Cycle 2

Negative control (N) with no added preservative

Positive control (P) with calcium propionate

Test Groups X, Y and Z with 1.5%, 0.75%, 3% of fermented flour and 0.75%, 1.25% and 0.375% of vinegar powder respectively (refer to Table 1 and 2.).

Table 1: Amount of ingredients used in Cycle 1

Negative Control Positive Control

A

B c

Note: This formulation is able to produce 4 loaves of each variation

Table 2: Amount of ingredients used in Cycle 2

Without any preservatives

Max permitted level of preservatives 1.5% diluted fermented flour + 0.75% diluted vinegar powder

0.75% diluted fermented flour + 1.25% diluted vinegar powder

3% diluted fermented flour + 0.375% diluted vinegar powder

Note: This formulation is able to produce 4 loaves of each variation Table 3: Amount of ingredients used in sensory evaluation

Variation: Negative Control

Variation: Positive Control

Variation: Test 1(A) Variation: Test 6(Z)

Note: This formulation is able to produce 02 loaves of each variation

Table 4: Formulation for the variations in Cycle 1 and Cycle 2

Note: The fermented flour or vinegar powder used in this study was diluted with plain flour inthe ratio ofl (FF/VP): 4 (flour). Table 5: Average Chewiness for different Variations

Table 5: Average Chewiness for different Variations

Methods:

Example 1: Bread making, packaging and storage

1.1: Dough preparation • Required ingredients were weighed.

• Ingredients (i.e. flour, yeast, salt, bread improver, preservatives) were dry mixedat low speed.

• Water was added after dry ingredients were well blended.

• The wet mass-incorporation was mixed for 3 minutes at low speed, changed to fast speed and continued mixing until the dough was smooth and extensible.

• Dough temperature was kept between (24-26) ±1°C during mixing.

1.2 Dough resting

• Dough was left to rest at room temperature for 10 minutes.

• It was then portioned into smaller doughs of 550g each, they were round andrested for 10 minutes at room temperature.

• Doughs were degased and shaped to fit the baking tin (Phoon Huat™, code: 1648 tin).

• Height of each dough before proofing was measured (readings were recorded).

1.3 Leavening

• The doughs were proofed at 30°C and 80% relative humidity until they reached aheight of (9.5 ± 0.5) cm, which is also about 80% height of the baking tin.

• Actual height after proofing and time taken to reach the height were noted (Readings were recorded).

1.4 Baking and cooling

• Baking tins were covered with a tin lid.

• Doughs were baked for 42 minutes at 240°C in a multi-deck oven.

• Loaves were cooled for 1 to 1.5 hours at room temperature until it was not warmto touch.

1.5 Volume scanning

• After cooling, volumes of loaves were analyzed by a volume scanner (PertenBVM 6630). Weight of loaves were taken as well (readings were recorded). 1.6 Packaging and storage

• Equipment in-contact with bread were sanitized before usage and avoided touching the bread with bare hands.

• Loaves were sliced to approximately 1.5cm thickness, end slices were discarded.

• Remaining bread slices were individually packaged into LDPE plastic bags.

• Before packing, the weight of each plastic bag was taken.

• After packing, the weight of bread was taken with the plastic bag (readings wererecorded).

• Plastic bags were sealed using an impulse heat sealer.

• All packs were labelled with their respective variations number, weight and date.

• Windowpane test was conducted to determine the readiness of the dough. Bread slices were incubated at 28 °C, 60% relative humidity.

Example 2: Fermented flour fermentation process inspection protocols

Example 2.1: A method for the determination of OD600 in fermentation broth

A method for the determination of acidi propioni at OD600 in fermentation broth was developed to provide a basis for the determination of OD600 by personnel in various departments. Briefly, the fermentation broth with acidi propioni was diluted with 0.5 mol/L HC1 by a factor of 0.5 to remove the effect of Ca (OH)2 from the fermentation broth, and the concentration of the bacteria, acidi propioni was measured by spectrophotometer at 600nm to control the absorbance range of 0-0.7. In order to prepare 0.5 mol/L HC1, 42 mL of concentrated hydrochloric acid was taken and volume was fixed to 1000 mL to make a 0.5 mol/L hydrochloric acid solution.

According to the fermentation time, an appropriate amount of the fermentation stock solution was drawn into two test tubes, then an appropriate amount of 0.5 mol/L HC1 was added to make appropriate dilutions and mixed on a vortex mixer. The absorbance of the fermentation dilutions was measured at 600nm using distilled water as a blank control. Further calculations were performed as below:

OD600=(OD600/l+OD600/2)/2*dilution times where:

OD600/1 - is the bacteriophage concentration of the fermentation dilution.

OD600/2 - is the bacteriophage concentration of the fermentation dilution.

OD600 - is the bacteriophage concentration of the original fermentation solution.

Example 2.3: A method for the determination of wet weight in fermentation broth

The wet weight of the bacterium in the fermentation broth is obtained by separating the bacterium from the fermentation broth by centrifugation and dilution with hydrochloric acid and discarding the supernatant. To obtain 2 mol/L HC1, 168 mL of concentrated hydrochloric acid is mixed to 1000 mL, i.e., 2 mol/L hydrochloric acid solution.

In order to determine the wet weight of the bacterium in the fermentation broth, two dry 10ml centrifuge tubes were prepared and marked as mil and m21, followed by pipetting 1 ml of fermentation broth sample into each of the two centrifuge tubes, and 9 ml of 2 mol/L HC1 into each of the tubes. The tubes were centrifuged at 5000 r/min for 20 min, supernatant was discarded, and tubes were weighed and named as ml2 and m22, followed by accumulation: m=[( ml2-ml l)+(m22-m21)]/2/0.001. where: ml2, m22 - weight after discarding supernatant, mi l, m21 - weight of the centrifuge tube. ml2, m22 - weight after discarding supernatant, mi l, m21 - weight of the centrifuge tube. Note: Two parallel runs of each sample were run together.

OD600 - is the bacteriophage concentration of the original fermentation solution. Example 2.4: Propionibacterium microscopy protocol

To detect the growth status of Propionibacterium in the fermentation broth and to validate whether the fermentation broth is contaminated with bacteria, a thin smear of the bacterial culture was taken over a clean slide by dipping an inoculating ring into a ring of fermentation solution and applying it to the centre of the slide.

The slide was dried over a high flame with slight heat or dry naturally followed by applying the smear face up and pass through the microfire 2-3 times. The slides were stained using crystalline violet staining as disclosed in the literature.

Example 2.5: Operating procedures for the determination of propionic acid content (HPLC method):

The water used for chromatography is in accordance with GB/T 6682 for Class I water and GB/T 6682 for Class III water for other water. The various reagent used for the purpose are Disodium hydrogen phosphate, Phosphoric acid, Acetonitrile, Propionic acid ((Vokai Chromatography Control GCS, >99.5%)), acetic acid (Vokai Chromatography Control GCS, >99.5%), lactic acid, (Aiko reagent DL-lactic acid, 85%, conversion required for calculation of results), succinic acid (Wokai chromatographic control GCS, >99.0%), butyric acid (n- butyric acid chromatographic control GCS, >99.5%), Dilute sulphuric acid: take 1.84 mol/L sulphuric acid 1 mL fixed volume 500 mL, that is, concentrated sulphuric acid dilution 5000 times.

Further, the standard mixed stock solution comprises 20g/L for propionic acid and lOg/L for lactic acid, acetic acid and succinic acid. 1g of propionic acid and 0.5g of each of lactic acid, acetic acid and succinic acid (to the nearest 0.0001g) were weighed accurately, dissolved and mixed with an appropriate amount of ultrapure water and fixed to 50mL to prepare a standard mixed stock solution with 20g/L of propionic acid and lOg/L of lactic acid, acetic acid and succinic acid. Reserve solution. Store at 4°C in a refrigerator for 3 months.

To prepare Butyric acid standard stock solution, about 0.25g of butyric acid (accurate to 0.0001g) was weighed, dissolved and mixed with an appropriate amount of ultrapure water, and fix the volume to 50mL to prepare a standard mixed stock solution with a butyric acid content of 5g/L. Store at 4°C in a refrigerator for 3 months. Microporous membranes: 0.45 pm and 0.22 pm, aqueous phase.

Example 2.6: High performance liquid chromatography conditions were kept as below Column: C18 column, 4.6mm><250mm, 5pm.

Mobile phase: 1.97% disodium hydrogen phosphate + 1% phosphoric acid + 2% acetonitrile, pH adjusted to 2.0 with phosphoric acid; filtered through a 0.45 pm microporous membrane in real time filtered through a 0.45 pm microporous membrane in vacuo.

Flow rate: 1.0 mL/min.

Column temperature: 35°C.

Injection volume: 20 pL.

Wavelength: 210 nm.

Reference condition for column cleaning: after the experiment, wash with 5% methanol for 30 min, then 50% methanol for 30 min, and finally with 100% methanol for 30 min. The column was cleaned with 100% methanol for 30 min. aqueous phase.

Example 2.7: Standard curve preparation

The final concentrations of the standard solutions were O.lg/L, 0.2g/L, 0.5g/L, Ig/L and 2g/L for propionic acid and 0.05g/L, O.lg/L and O.lg/L for lactic acid, acetic acid and succinic acid respectively. 0. Ig/L, 0.25g/L, 0.5g/L, Ig/L, filtered through a 0.22pm microporous membrane, and injected into the sample from low to high concentration, with the concentration as the horizontal coordinate and the peak area as the vertical coordinate, to plot the standard curve.

The final concentrations of butyric acid standard solutions were O.Olg/L, 0.04g/L, 0.08g/L, O.lg/L and 0.4g/L, respectively, and were filtered through a 0.22pm microporous membrane and the samples were injected from the lowest to the highest concentration. The standard curve was plotted with the concentration as the horizontal coordinate and the peak area as the vertical coordinate. aqueous phase.

Example 2.7: Sample determination

About 1.0 ml (to the nearest 0.0001g) of the sample was dissolve in water and volume was fixed to 100 mL. Out of the stock solution, 1 mL of the sample solution was diluted with dilute sulphuric acid to 10 mL, i.e. dilute it 10 times, filter it through a 0.22 pm microporous membrane and inject it into the sample for determination.

The propionic acid (acetic acid, lactic acid, succinic acid, butyric acid) content of the specimen is calculated according to equation (1).

Xl= cxn/mxl00% (1) where:

XI -content of propionic acid (acetic, lactic, succinic, butyric) in the sample, %.c- concentration of propionic acid in the sample solution derived from the standard curve, in g/L; n- number of dilutions m - mass of sample per litre of sample solution, g. Two significant figures are retained for the calculation.

Example 3: Data manipulation for day of baking

Based on the above recorded results, height increased during proofing, rate of fermentation for variations by estimating time to attain 80% of their height increment and specific volume of bread produced were obtained through the following formulas: height after proofing-height before proofing Height increased during proofing (%) = height before proofing

Estimated time to attain 80% height increment (min) = [(1)/ T]* 80 (2), where T is the time taken for bread dough to rise to its actual height after proofing. Specific volume (ml/g) = volume of loaf after cooling weight of loaf after cooling

3.1: Test conducted over the 15-day

For this shelf-life study, analytical and microbial analyses took place over a 15-dayperiod for each cycle, following the day of baking (Day 0). The bread slices were categorized into two groups. In the first group, fifteen bread slices from each variation were taken for the visual inspection of the mould growth over the 15-day period. Thesecond group were the remaining bread slices, intended for analytical and microbiological testing use. For this study, visual inspection was done daily whereas analytical tests and microbial tests were done on Odd days (i.e., Day 1, 3, 5, 7, 9, 11,13 and 15 (Fig 1).

3.2 Moisture loss

Weight of bread slices was subsequently measured again with the plastic bags on alternate days over the 15-day period. Moisture loss of bread slices were determined using the following formula:

Moisture loss (%) = Weight of bread with plastic bag on Day X~ Weight of plastic bag on Day 0 Weight of bread with plastic bag on Day 10 whereby B was also the densest. Due to technical issues, Day 7 results fluctuate considerably. Hence, it was withdrawn. From the graph, hardness level increased tremendously from Day 1 to Day 3, indicating sensory deterioration due to stalling. Since there was moisture loss during storage, the hardness of bread increased with time (Fig.12).

Interestingly, springiness of bread had decreased over time, indicating the loss of ability to return to its original shape after a force was being applied. In relation to Fig. 12 (b), chewiness wasdirectly proportional to hardness level as the harder the bread gets, the chewier it will be (Fig. 13). Despite X and Y not meeting an 80% height increase during proofing, they managed to attain specific volume slightly higher than N or close to P. Hence, the addition of vinegar powder or reduction in fermented flour has helped in enhancing the loaf volume. Thus, producing a lighter bread (Fig.17).

3.3 Effect of preservatives on fermentation and loaf volume

Table 6. Measurements obtained on the day of baking for Cycle 2 variations

It was observed with reference to this graph, Y that contains 0.75% fermented flour and 1.25% vinegar powder took the shortest time followed by N, X, Z and P. According to the increasing amount of fermented flour/decreasing amount of vinegar powder (Z>X>Y), the proofingtime of the dough was longer (Fig.14).

The height before and after proofing varied between variations. Hence, not all variationhad an 80% height increased during proofing. Even though the actual proofing time for Y in Fig. 14 was similar to N, their percentage height increase was different by 14.89%. Whereby N increased by 73.66%, Y only increased by 58.77%. In general, X, Y and Z hada lower height increase (Fig.15). The rate of fermentation was the fastest in Variation N that contains no preservative as ithad proved to desire height in the shortest amount of time, followed by Y, X/ P and Z. InCycle 1, the estimated time of P was longer than A (3% fermented flour). However, in Cycle 2, Z was longer than P, showing that vinegar powder does have a yeast inhibitory property (Fig.16).

Despite X and Y not meeting an 80% height increase during proofing, they managed to attain specific volume slightly higher than N or close to P. Hence, the addition of vinegar powder or reduction in fermented flour has helped in enhancing the loaf volume. Thus, producing a lighter bread (Fig.17).

Example 3.4: Effect of preservatives on shelf-life stability

Mould was observed the earliest for N on Day 3. X and Y started moulding on Day 5 while P began on Day 7. Z only started moulding on Day 11. At the end of the 15 -day shelf-life study, N and X had all grown mould, P and Y each left with 1 slice of bread without moulds, while Z still had 9 slices left. Thus, showing Z was more shelf stable as compared to the rest. In comparison with Cycle 1, the combined effect of vinegar powder (0.375%) and fermented flour (3%) in shelf life stability was better than fermented flour alone (3%) (Fig.18).

Table 7. Microbiological analysis (NA) performed on Odd days for Cycle 2 From the above table, P had first exceeded the microbial limit on Day 9; X on Day 13;

Yon Day 13 and Z on Day 9.

Table 8. Microbiological analysis (PDA) performed on odd days for Cycle 2

Days with (-) indicates no microbial test was performed due to shortage of bread samples. Plates that were unable to count due to inadequate sample dilution (TNTC) or contamination were denoted with (*). Colony-forming units per gram of bread sample exceeding 1 x 10 ² cfu/g for yeast and mould (PDA) are deemed as unsatisfactory for consumption.

Microbial limit for P first exceeded on Day 7 while Day 5 for X. Y exceeded on Day 9 while Z on Day 11. For microbial testing, some of the results did not tally with the visualinspection of mould growth. For Sample Y, mould first appeared on Day 5. However, yeast and mould count were Ocfu/g from Day 5 to Day 7. The unexpected result was probably caused by the different moulding rate of bread slices. The slices that were picked at random for microbial testing might be free of yeast and mould. Despite X and Y not meeting an 80% height increase during proofing, they managed toattain specific volume slightly higher than N or close to P.

Hence, the addition of vinegar powder or reduction in fermented flour has helped in enhancing the loaf volume. Thus, producing a lighter bread (Fig.17). Possible factors affecting shelf-life stability:

The pH on Day 1 was significantly different between variations. X and Y that contain ahigher dosage of vinegar powder had a lower pH value of 5.45 and 5.32 respectively. Due to their low acid property and a lower amount of fermented flour being used, dough of X and Y were slightly more extensible. Despite the low percentage increase inheight during proofing, they could expand and rise to desired volume during baking. Therefore, vinegar powder enhances loaf volume. With regard to shelf life, vinegar powder does not lower the pH of bread significantly. Hence, pH is not a factor that enhances its shelf life (Fig.19).

In general, the water activity of bread produced in Cycle 2 was between 0.950 and 0.955. On Day 1, water activity of X, Y and Z was slightly higher than P and N. Throughout thecycle, water activity decreased gradually as it was used for microbial activity and moisture loss during prolonged storage (Fig.20).

Moisture content of N was significantly lower than the other variations on Day 1. Overall, the test groups X, Y, Z had higher moisture content than N and P. Hence, thisindicated that the combination of fermented flour and vinegar powder had improved the water-binding capacity of dough during mixing, lowering the loss of moisture during dough preparation. Throughout the cycle, the water content of bread decreasedsteadily due to loss of moisture during storage (Fig.21a and 21b).

Effect of preservatives in retaining moisture of bread during storage

A high linear correlation between moisture loss and time was determined (R ² > 0.9). The moisture loss between variations was about the same. There was no relationship between the added vinegar powder and its water retention property observed. This interpretation was based on the fact that all LDPE plastic bags have the same vapor permeability (Fig.22).

Effect of preservatives on bread quality

The L* value of X is significantly higher than the rest on Day 1. Therefore, it was lighterin colour as compared to other variations. In general, the values for L*, a* and b*between variations and throughout the shelf-life study were very close. Hence, therewas not much difference in terms of colour regardless of the formulations (Fig. 23).

Hardness level of bread on Day 1 was not significantly different from each other. In Cycle 1, formulations that contain fermented flour (3%, 4%, 5%) were significantly harder than the control variations. However, in Cycle 2, no significant difference in hardness was detected between X/Y/Z and N/P even though X/Y/Z contains fermented, flour. Therefore, the addition of vinegar powder improved the texture of bread, makingit softer. This implication can be related to specific volume whereby N; P, X and Y had asimilar value, they were of similar lightness. Throughout the cycle, the hardness level increased for all variations. This was resulted from the loss of moisture during storage.

On Day 1, both springiness and chewiness of all variations were not significantly different. This suggested that the combination of fermented flour and vinegar powder does not affect these two texture attributes on Day 1. The springiness level of the bread decreased drastically over the days. As seen in Fig. 25(a), the springiness level of P and Xwere comparable since the springiness level of both drops at a similar rate. The chewiness level of the bread increased significantly throughout the cycle. The chewiness level of X and Z was higher than P, particularly during the earlier stage of the cycle.

Since chewiness corresponds directly to hardness level, this suggested that the addition of fermented flour or vinegar powder to breads had increased their susceptibility tostaling. Hence, they were harder and chewier than P during prolonged storage (Fig. 25).

Sensory Evaluation

The mean results obtained for the bread analyzed were shown in Fig. 26. The analysis ofthe mean scores given by the panelists for each bread variation with ANOVA (Two-Factor without replication) and Duncan test revealed the significant differences forthese seven attributes. For the appearance, moistness, softness and taste attribute, the mean value of N was significantly higher than the other three bread variations. This implied that the panelists least like N among all the variations in terms of these attributes. For the aroma attributes, N was significantly higher than Z in terms of their mean values. For the chewiness attribute, there was no significant difference between thevariations, which means the consumers’ acceptability were the same for all the variations. Overall, N was significantly lower than A and Z in the mean values. This concluded that A and Z were better liked by the panelists as a whole. P, A and Z were not significantlydifferent from each other. Thus, A and Z were comparable to P in terms of consumers ‘acceptability. In a nutshell, fermented flour at 3% or the combination with 0.375% vinegar powder can produce bread of similar sensory quality as a commercial breadmade of calcium propionate (Fig. 26).

Survey finding:

Responses from the 26 respondents that purchase bread when visiting the supermarketwere reviewed. When asked for the ideal shelf life of a bread, 17 respondents (65%) selected “3-7 days”, 8 respondents (31%) selected “1-2 weeks”, 1 respondent (4%) selected “2-3 weeks”, and none selected “more than 3 weeks”. Overall, the majority viewed “3-7 days” as the ideal shelf life of a bread (Fig. 27). When asked for their willingness to pay more for bread using natural preservatives instead of chemical preservatives, 6 (23%) selected “5-10%”, 9 (35%) selected “10-15%”, 10 (38%) selected “15-20%”, while 1 (4%) selected “not willing to purchase”. An estimateof one-third each was willing to pay for 5-10% and 20-15% increased from original cost of a bread (Fig. 28).

Shelf life, cost and health are not significantly different in terms of the mean for the respondent’s concern at 5% significance level. Texture/taste has a lower mean and is significantly lower than shelf life, cost and health at 5% significance level. This can be inferred that the respondents’ priorities texture/taste over the other aspect.

Advantages of the embodiments:

1. The fermented flour disclosed in the present embodiments is preservative free.

2. The fermented flour disclosed in the present embodiments have a higher shelf life.

3. The fermented flour disclosed in the present embodiments have appropriate time of proofing.

4. The fermented flour disclosed in the present embodiments has an appropriate pH as required for suitable baking.

5. The fermented flour disclosed in the present embodiments is cost effective.

In the application, unless specified otherwise, the terms "comprising", "comprise", and grammatical variants thereof, intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, non-explicitly recited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

It will be apparent that various other modifications and adaptations of the application will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the application and it is intended that all such modifications and adaptations come within the scope of the appended claims.