WO2019058248A1 - Yeast strain usable for reducing the quantity of acrylamide in a thermally treated food - Google Patents

Yeast strain usable for reducing the quantity of acrylamide in a thermally treated food Download PDF

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Publication number
WO2019058248A1
WO2019058248A1 PCT/IB2018/057130 IB2018057130W WO2019058248A1 WO 2019058248 A1 WO2019058248 A1 WO 2019058248A1 IB 2018057130 W IB2018057130 W IB 2018057130W WO 2019058248 A1 WO2019058248 A1 WO 2019058248A1
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Prior art keywords
strain
food
aureobasidium pullulans
potato
acrylamide
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PCT/IB2018/057130
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French (fr)
Inventor
Marta MARI
Alessandra DI FRANCESCO
Luisa UGOLINI
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Alma Mater Studiorum - Universita' Di Bologna
Consiglio Per La Ricerca In Agricoltura E L'analisi Dell'economia Agraria
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Publication of WO2019058248A1 publication Critical patent/WO2019058248A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/10—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops
    • A23L19/12—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops of potatoes
    • A23L19/18—Roasted or fried products, e.g. snacks or chips
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • Yeast strain usable for reducing the quantity of acrylamide in a thermally treated food
  • the invention relates to a yeast strain and the use of the latter to treat foods, in particular foods of vegetable origin, such as for example potatoes, so as to reduce the amount of acrylamide that is produced in the aforesaid foods because of a thermal treatment, in particular frying.
  • the invention can be applied industrially in the field of the transformation of foods, in particular in the transformation of foods of vegetable origin and more in particular in the potato industry.
  • the acrylamide (2-propenamide; acrylic acid amide) is a chemical substance that forms naturally and in various food products (for example potatoes) because of chemical reactions induced by high temperature thermal treatments (for example frying) to which the aforesaid products are subjected.
  • the acrylamide was identified for the first time in 2002 and in 2007 the European Food Safety Authority (EFSA) produced a first evaluation of the risks arising from the presence of acrylamide in food products.
  • EFSA European Food Safety Authority
  • the conclusions of EFSA are that, in consumers of all age groups, the acrylamide can increase the risk of developing a cancer.
  • EFSA has therefore set an admissible maximum limit for acrylamide in (thermally treated) food products, which limit is 0.6 mg/kg for fried potatoes.
  • the latter comprise the stick-shaped fried potatoes (so-called "French fries” or “fries”) and the fried potatoes having the shape of thin and crunchy slices (so-called "chips" or "crisps").
  • the chemical process that leads to the formation of acrylamide is known as the "Maillard reaction”.
  • the latter is a complex system of reactions, which can be nevertheless schematized as a condensation reaction between some sugars and an amino acid, in particular asparagine, which are normally contained in many foods, such as for example potatoes.
  • the Maillard reaction can in general be considered to be a substantially “useful” reaction for the food industry, because it enables the food products to be toasted and to make the latter tastier and more appetizing (also visually) for a consumer.
  • the "blanching" is carried out, inter alia, to favour an extraction of the chemical precursors of the acrylamide from the potatoes.
  • the immersion in acid solution is carried out for the purpose of lowering the pH of the food matrix, it being known that the Maillard reaction is affected by the pH values (Chemistry and Safety of Acrylamide in Food, by: M. Friedman, D. Mottram; Springer- Verlag, 2005).
  • a lowering of the pH - caused by the addition of an acidulant (citric acid) to the food matrix - converts the free non protonated amines into protonated amines, which could block the reaction of formation of the acrylamide (Jung, M.Y., Choi, D.S., Ju, J.W. A novel technique for limitation of acrylamide formation in fried and baked com chips and in French fries. Journal of Food Science, 68:1287-1290, 2003).
  • a drawback that is found in both "blanching" and immersion in acid solution is that these methods not only produce an effect on the level of acrylamide in the finished product (fried potato), but can also affect the organoleptic features (for example aroma, colour, texture) of the latter.
  • the enzyme pre-treatment method provides for treating the potatoes with asparaginase, i.e. with an enzyme that catalyzes asparagine hydrolysis into ammonia and aspartic acid.
  • a drawback of this method consists of the significant financial onerousness thereof.
  • An object of the invention is to improve the known methods for reducing the formation of acrylamide in thermally treated foods.
  • Another object is to improve the known methods for reducing the formation of acrylamide in thermally treated food of vegetable origin.
  • a further object is to improve the known methods for reducing the formation of acrylamide in thermally treated potatoes and in particular in fried potatoes.
  • a further other object is to make available a method for reducing the formation of acrylamide in fried potatoes that does not influence the organoleptic features of the latter.
  • Still another object is to make available a method for reducing the formation of acrylamide in fried potatoes that is substantially inexpensive.
  • a still further other object is to make available a method for reducing the formation of acrylamide in fried organic potatoes, such as to produce a low environmental impact and maintain substantially unaltered the properties of the aforesaid organic potatoes.
  • a method using a yeast strain is provided, as defined in claim 1.
  • a use of a yeast strain is provided, as defined in claim 19.
  • composition comprising a yeast strain is provided, as defined in claim 26.
  • a yeast strain Owing to these aspects, a yeast strain, a composition incorporating the aforesaid strain and a method are provided that enable to reduce the formation of acrylamide in a food, in particular a vegetable food and more in particular potatoes, which is thermally treated at temperatures that are suitable for frying, without influencing the organoleptic features of the food and in a substantially inexpensive manner.
  • the invention enables the formation of acrylamide in fried organic potatoes to be reduced and at the same time it enables the properties of the latter to be maintained substantially unaltered.
  • the strain, the composition and the method according to the invention enable the use of drastic physical and/or chemical treatments to be avoided.
  • the above-mentioned yeast strain is the LI strain of Aureobasidium pullulans, which has been identified during periodic screenings of the microflora present on the surface of leaves and fruits of cultivated plants. These screenings had been conducted in order to identify effective means for fighting plant diseases.
  • Figure 1 is a photograph obtained by SEM (Scanning Electron Microscope) of the strain according to the invention, which was grown on a peach;
  • Figure 2 shows a sequence of ribosomal DNA of the strain according to the invention, compared with a corresponding sequence of a reference strain
  • Figure 3 shows an HPLC chromatogram obtained from a homogenized vegetable food sample, in the absence of the strain according to the invention
  • Figure 4 shows an HPLC chromatogram obtained from a homogenized vegetable food sample, in the presence of the strain according to the invention
  • Figure 5 is a photograph of a sample of fried potatoes of "chips" type that have not been treated with the strain according to the invention.
  • Figure 6 is a photograph of a sample of fried potatoes of "chips" type that have been treated with the strain according to the invention.
  • treating thermally a raw food, in particular a raw food of vegetable origin and more in particular a - raw potato, at temperatures that are suitable for frying means subjecting the aforesaid potato to temperatures that are such as to transform the raw potato into a fried potato.
  • the temperatures that are suitable for frying are normally comprised between 160°C and 190°C. This temperature range is nevertheless not to be considered limiting, because the person skilled in the art is conscious of the fact that the potatoes can be fried at temperatures below 160°C and above 190°C.
  • the term "food” defines any transformed, partially transformed or non transformed substance or product, intended to be swallowed, or which can be reasonably expected to be swallowed, by human beings.
  • Aureobasidium pullulans (De Bary) Araaud is commonly known as "black yeast” and it has been given many names over the last 90 years because of the variety of environments in which it can develop.
  • Table 1 shows the scientific classification of this yeast:
  • Aureobasidium sp. is comprised in the epiphyte microbial flora of fruits and leaves of typical plants of the temperate regions.
  • the use of Aureobasidium sp. as a biological control agent against fungal pathogens of fruits, in particular in the post-harvest phase gave rise to a certain interest.
  • yeast or yeast-like fungi are particularly interesting for various reasons.
  • the biological activity of these microorganisms does not derive from the production of antibiotic substances, as on the other hand occurs with many bacteria.
  • these microorganisms do not have negative impacts from an environmental and toxicological point of view.
  • these microorganisms are easily cultivatable on a large scale, through the use of inexpensive and easily available substrates.
  • the strain according to the invention - namely the LI strain of A. pullulans - was isolated from the surface of "Redhaven" cultivar peaches grown in a peach orchard of the University of Bologna located in Cadriano (Man M., Martini C, Guidarelli M., Neri F., 2012. Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Biol Control 60: 132- 140). The strain was selected among different microorganisms in a study on identifying microbial antagonists.
  • the LI strain was deposited on date 22 March 2017 at DBVPG (Industrial Yeasts Collection, Borgo XX Giugno, 74, 06121 Perugia, Italy) with number 39P.
  • the LI strain is grown in laboratory on an NYDA agar substrate and the optimum growth temperature is 25°C.
  • the NYDA agarized substrate is produced by dissolving the following ingredients in 1000 ml of distilled water: 8 grams of Nutrient Broth, 5 grams of Yeast Extract, 10 grams of dextrose and 15 grams of agar.
  • the LI strain has a strong attitude in resisting the development of some fruit phytopathogenic fungi, exhibiting a reduction of the disease greater than 50%.
  • Figure 1 shows yeast-like cells of the LI strain of A. pullulans, photographed under the Scanning Electron Microscope (SEM), which cells were grown on a peach of the aforesaid "Redhaven” cultivar.
  • SEM Scanning Electron Microscope
  • Figure 2 shows the 100% homology between the ITS sequence of the LI strain of A. pullulans and a corresponding ITS sequence of the CBS 584.75T strain of A. pullulans.
  • the ITS sequence of the LI strain of A. pullulans was further disclosed in a sequence listing (drawn up in compliance with WIPO Standard ST.25), which is attached to the present patent application and in which the aforesaid ITS sequence is named as SEQ ID NO: 1.
  • the LI strain has shown a certain effectiveness in reducing the occurrence of rots on fruits after harvesting, namely during the phase of conservation and marketing.
  • the application of the LI strain on the fruits has reduced vegetable pathologies such as grey mould, brown rot and green-blue mould on apples, actinidia and oranges.
  • the Inventors discovered the ability to metabolize the asparagine exhibited by the LI strain.
  • the LI strain used asparagine as a nutrient, producing a resulting accumulation of aspartic acid in the substrate (5% peach juice inoculated with the LI strain).
  • the Inventors decided to extend the experimental investigation to the potatoes.
  • the asparagine is in fact one of the main amino acids contained in the potatoes and is (as highlighted previously) an important precursor of the acrylamide.
  • the problem is known of the formation of acrylamide in potatoes following high temperature thermal treatment (frying)
  • the Inventors evaluated the possible ability of the LI strain to reduce the asparagine content in the potatoes in order to reduce the formation of acrylamide in transformed potatoes, such as fried potatoes of "French fries" type (stick-shaped) and fried potatoes of "chips” type (thin and crisp slices).
  • Example 1 a preliminary experimental test is disclosed that was conducted on a potato-based preparation.
  • Example 1 Reduction of the asparagine content in a potato-based preparation by contact with LI strain of Aureobasidium pullulans
  • a homogenized potato product was prepared, by using a homogenizer of known type.
  • a suspension in distilled water of the LI strain of Aureobasidium pullulans was prepared having a concentration (of yeast cells) of 10 8 CFU/ml.
  • the aforesaid suspension is an example of a liquid formulation of the composition according to the invention.
  • the suspension can be prepared by using tap water instead of distilled water. This liquid formulation is conservable stably (i.e. maintainable alive) for a maximum time of 120 hours at a temperature of 4 °C.
  • a part of the homogenized potato product was placed in the LI strain suspension, by using an amount of homogenized potato product and of suspension such as to obtain a 1 : 1 (w/v) ratio. This sample was called "LI".
  • a remaining part of the homogenized potato product (quantitatively equal to the LI sample) was not placed in contact with the LI strain suspension, but with an equal volume of water.
  • This control sample was called "Test”.
  • the homogenized potato product was left in contact with the LI strain suspension for a time (contact time) of 30 minutes and at a temperature of 25°C.
  • the Test sample (homogenized potato product, without LI strain) was left for 30 minutes at a temperature of 25°C. After the aforesaid contact time elapsed, the extraction and analysis of free amino acids were made.
  • the samples were diluted with water in a 1 :5 (w/v) ratio, subjected to ultrasound for 20 minutes and the liquid phase extract was recovered by centrifugation and was subsequently filtered (0.22 ⁇ ) to eliminate the yeast.
  • the peaks in the chromatograms are indicated as: 1, aspartic acid; 2, glutamic acid; 3, asparagine; 4, serine; 5, glutamine; 6, threonine; 7, alanine; 8, arginine; 9 valine; 10, isoleucine/ phenylalanine; 11, leucine/lysine; SI, internal standard.
  • the analysis of the Test sample ( Figure 3) highlighted the peak of the asparagine (peak 3, in grey), which is on the other hand absent in the - chromatogram obtained from the analysis of the potato sample that was in contact with the LI strain.
  • Example 1 The results obtained in the Example 1 confirmed the ability of the LI strain of Aureobasidium pullulans to reduce the amount of asparagine contained in a substrate that is different from a vegetable juice (peach juice), such as a potato-based preparation (homogenized potato and water product).
  • peach juice such as a potato-based preparation (homogenized potato and water product).
  • the strain according to the invention significantly metabolized the asparagine (which decreased from 652.0 mg/kg in the Test sample to less than 4.5 mg/kg in the LI sample, a value at the limit of detectability of the quantitative analysis by HPLC), causing a consequent increase in the aspartic acid (which increased from 395.4 mg/kg in the Test sample to 988.7 mg/kg in the LI sample).
  • Example 2 Reduction of the acrylamide content in fried potatoes of "chips" type by treatment with the LI strain of Aureobasidium pullulans before the frying step.
  • the potatoes were peeled, cut so as to obtain thin slices measuring about 1 mm and divided into two batches.
  • One batch was immersed for 30 minutes (contact time) in a suspension of the LI strain of Aureobasidium pullulans in distilled water in a 3 x 10 6 CFU/ml concentration (sample treated with LI strain), in a 1 :2 w/v ratio.
  • the aforesaid suspension constitutes an example of a liquid formulation of the composition according to the invention.
  • the suspension can be prepared by using tap water instead of distilled water.
  • Figure 5 is a photograph of the untreated sample after frying
  • Figure 6 is a photograph of the sample treated with LI strain after frying. Comparing Figure 5 with Figure 6 it can be clearly noted that, in the untreated sample, during the frying step the Maillard reaction took place more intensely that in the sample treated with the LI strain, as the slices of Figure 5 appear darker (i.e. more browned) than the slices of Figure 6. This organoleptic element is considerable as a substantial confirmation of the analytical results of Table 2.
  • Example 2 the composition according to the invention is disclosed as a liquid suspension of LI strain of A. pullulans, to a person skilled in the art the possibility appears clear of applying known methods (like those used, for example, in oenology and in the bakery products industry) to dehydrate the aforesaid suspension, so as to obtain a composition containing up to 95 - 97% of solid substance.
  • the strain according to the invention is vital, but remains metabolically inert until rehydration of the composition.
  • the solid formulation of the composition according to the invention can be conserved and maintained stable for about 12-14 months.
  • Example 2 the potatoes have been so cut as to obtain thin slices ("chips” or “crisps"), it is possible to treat with the strain, the composition and the method according to the invention also differently shaped potatoes, for example potatoes cut into the shape of a stick ("French fries” or “fries”).
  • a suspension of LI strain of Aureobasidium pullulans in distilled water was used in a 3 x 10 6 CFU/ml concentration, it is reasonable to state that the LI strain is also usable in a lower concentration, for example a 1 x 10 6 CFU/ml concentration (i.e. 10 6 CFU/ml).
  • the ability of the LI strain of Aureobasidium pullulans to metabolize the asparagine was detected experimentally for the first time in a growth substrate different from potatoes. It can thus be hypothesized that the strain, the composition and the method according to the invention can be effectively used to reduce the amount of asparagine contained in foods and food matrices that are different from potatoes. In this manner, the strain, the composition and the method according to the invention could be used to reduce the amount of acrylamide that forms in foods and food matrices that are different from potatoes due to a thermal treatment at high temperatures, for example above 140°C.

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Abstract

A method for reducing the amount of asparagine that is contained in a raw food, so as to reduce the amount of acrylamide that forms in said food when said food is thermally treated at temperatures that are suitable for frying or cooking, comprises treating said raw food with the L1 strain of Aureobasidium pullulans that is able to metabolize the asparagine and deposited with number 39P. A composition, comprising the aforesaid L1 strain of Aureobasidium pullulans, is usable for reducing the amount of asparagine that is contained in a raw food, so as to reduce the amount of acrylamide that forms in said food when said food is thermally treated at temperatures that are suitable for frying or cooking.

Description

Yeast strain usable for reducing the quantity of acrylamide in a thermally treated food
■ [0001 ] The invention relates to a yeast strain and the use of the latter to treat foods, in particular foods of vegetable origin, such as for example potatoes, so as to reduce the amount of acrylamide that is produced in the aforesaid foods because of a thermal treatment, in particular frying.
[0002] The invention can be applied industrially in the field of the transformation of foods, in particular in the transformation of foods of vegetable origin and more in particular in the potato industry.
[0003] The acrylamide (2-propenamide; acrylic acid amide) is a chemical substance that forms naturally and in various food products (for example potatoes) because of chemical reactions induced by high temperature thermal treatments (for example frying) to which the aforesaid products are subjected. The acrylamide was identified for the first time in 2002 and in 2007 the European Food Safety Authority (EFSA) produced a first evaluation of the risks arising from the presence of acrylamide in food products. The conclusions of EFSA are that, in consumers of all age groups, the acrylamide can increase the risk of developing a cancer. EFSA has therefore set an admissible maximum limit for acrylamide in (thermally treated) food products, which limit is 0.6 mg/kg for fried potatoes. Typically, the latter comprise the stick-shaped fried potatoes (so-called "French fries" or "fries") and the fried potatoes having the shape of thin and crunchy slices (so-called "chips" or "crisps").
[0004] The chemical process that leads to the formation of acrylamide is known as the "Maillard reaction". The latter is a complex system of reactions, which can be nevertheless schematized as a condensation reaction between some sugars and an amino acid, in particular asparagine, which are normally contained in many foods, such as for example potatoes. It should be noted that, notwithstanding the problem of the formation of the acrylamide, the Maillard reaction can in general be considered to be a substantially "useful" reaction for the food industry, because it enables the food products to be toasted and to make the latter tastier and more appetizing (also visually) for a consumer.
[0005] Despite the numerous researches conducted for the purpose of understanding fully the mechanisms of formation of the acrylamide, it has not yet been possible to draw up effective guidelines that are suitable for minimizing the formation of the aforesaid chemical substance in thermally treated food products. [0006] A need that is felt particularly by the food industry is thus to produce fried potatoes that have a low acrylamide content. For this purpose, some methods for pre- ■ treating the potatoes before the frying step have been proposed and are currently used by the food industry in order to attempt to reduce the subsequent formation of acrylamide. Known methods comprise, for example, the so called "blanching", the immersion in acid solution and the enzyme pre-treatment.
[0007] The "blanching" (scalding) is carried out, inter alia, to favour an extraction of the chemical precursors of the acrylamide from the potatoes. The immersion in acid solution is carried out for the purpose of lowering the pH of the food matrix, it being known that the Maillard reaction is affected by the pH values (Chemistry and Safety of Acrylamide in Food, by: M. Friedman, D. Mottram; Springer- Verlag, 2005). In particular, a lowering of the pH - caused by the addition of an acidulant (citric acid) to the food matrix - converts the free non protonated amines into protonated amines, which could block the reaction of formation of the acrylamide (Jung, M.Y., Choi, D.S., Ju, J.W. A novel technique for limitation of acrylamide formation in fried and baked com chips and in French fries. Journal of Food Science, 68:1287-1290, 2003).
[0008] A drawback that is found in both "blanching" and immersion in acid solution is that these methods not only produce an effect on the level of acrylamide in the finished product (fried potato), but can also affect the organoleptic features (for example aroma, colour, texture) of the latter.
[0009] The enzyme pre-treatment method provides for treating the potatoes with asparaginase, i.e. with an enzyme that catalyzes asparagine hydrolysis into ammonia and aspartic acid. A drawback of this method consists of the significant financial onerousness thereof.
[0010] An object of the invention is to improve the known methods for reducing the formation of acrylamide in thermally treated foods.
[001 1] Another object is to improve the known methods for reducing the formation of acrylamide in thermally treated food of vegetable origin.
[0012] A further object is to improve the known methods for reducing the formation of acrylamide in thermally treated potatoes and in particular in fried potatoes.
[0013] A further other object is to make available a method for reducing the formation of acrylamide in fried potatoes that does not influence the organoleptic features of the latter. [0014] Still another object is to make available a method for reducing the formation of acrylamide in fried potatoes that is substantially inexpensive.
[0015] A still further other object is to make available a method for reducing the formation of acrylamide in fried organic potatoes, such as to produce a low environmental impact and maintain substantially unaltered the properties of the aforesaid organic potatoes.
[0016] In a first aspect of the invention, a method using a yeast strain is provided, as defined in claim 1.
[0017] In a second aspect of the invention a use of a yeast strain is provided, as defined in claim 19.
[0018] In a third aspect of the invention a composition comprising a yeast strain is provided, as defined in claim 26.
[0019] Owing to these aspects, a yeast strain, a composition incorporating the aforesaid strain and a method are provided that enable to reduce the formation of acrylamide in a food, in particular a vegetable food and more in particular potatoes, which is thermally treated at temperatures that are suitable for frying, without influencing the organoleptic features of the food and in a substantially inexpensive manner. In particular, the invention enables the formation of acrylamide in fried organic potatoes to be reduced and at the same time it enables the properties of the latter to be maintained substantially unaltered. In fact, unlike the known methods, the strain, the composition and the method according to the invention enable the use of drastic physical and/or chemical treatments to be avoided.
[0020] The above-mentioned yeast strain is the LI strain of Aureobasidium pullulans, which has been identified during periodic screenings of the microflora present on the surface of leaves and fruits of cultivated plants. These screenings had been conducted in order to identify effective means for fighting plant diseases.
[0021] The use of the LI strain of Aureobasidium pullulans to reduce acrylamide in thermally treated vegetable products, in particular in the fried potatoes, is based on a surprisingly unexpected technical effect, i.e. the ability of the aforesaid microorganism to metabolize the asparagine contained in a substrate and to reduce significantly the concentration thereof in the substrate. The mode by which the aforesaid unexpected technical effect was discovered by the Inventors will be illustrated with greater detail below. [0022] Owing to the aforesaid LI strain, when the substrate (for example, a raw potato) is subsequently thermally treated (for example, subjected to frying), it is possible to reduce » significantly the formation of, and the resulting contamination by, acrylamide without the use of particular chemical substances, but through a natural and low-impact process. The invention thus enables the use of chemical substances (for example, acid solutions) and/or physical treatments (for example, scalding) to be limited in order to remove the asparagine from the potatoes, so as not to alter the organoleptic properties of the food product. Moreover, whilst the use of asparaginase represents a significant burden on a financial point of view, treating the potatoes (before frying) through the method according to the invention enables costs to be reduced significantly, which is particularly important for the food industry.
[0023] The invention can be better understood and implemented with reference to the enclosed drawings that illustrate an embodiment thereof by way of non-limiting example, in which:
[0024] Figure 1 is a photograph obtained by SEM (Scanning Electron Microscope) of the strain according to the invention, which was grown on a peach;
[0025] Figure 2 shows a sequence of ribosomal DNA of the strain according to the invention, compared with a corresponding sequence of a reference strain;
[0026] Figure 3 shows an HPLC chromatogram obtained from a homogenized vegetable food sample, in the absence of the strain according to the invention;
[0027] Figure 4 shows an HPLC chromatogram obtained from a homogenized vegetable food sample, in the presence of the strain according to the invention;
[0028] Figure 5 is a photograph of a sample of fried potatoes of "chips" type that have not been treated with the strain according to the invention;
[0029] Figure 6 is a photograph of a sample of fried potatoes of "chips" type that have been treated with the strain according to the invention.
[0030] In the context of the present description and the attached claims, the use of the term "potato" in the singular does not rule out that the strain, the composition and/or the method according to the invention are able to provide for the desired technical effect, i.e. the reduction of the asparagine content, when used to treat a plurality of potatoes.
[0031] In the context of the present description and the attached claims, the terms "Aureobasidium pullulatis" and "A. pullulatis" are considered to be synonyms and are thus used in a mutually interchangeable manner. [0032] In the context of the present description and the attached claims, treating thermally a raw food, in particular a raw food of vegetable origin and more in particular a - raw potato, at temperatures that are suitable for frying means subjecting the aforesaid potato to temperatures that are such as to transform the raw potato into a fried potato. For a person skilled in the art, the temperatures that are suitable for frying are normally comprised between 160°C and 190°C. This temperature range is nevertheless not to be considered limiting, because the person skilled in the art is conscious of the fact that the potatoes can be fried at temperatures below 160°C and above 190°C.
[0033] In the context of the present description and the attached claims, the term "food" defines any transformed, partially transformed or non transformed substance or product, intended to be swallowed, or which can be reasonably expected to be swallowed, by human beings.
[0034] Aureobasidium pullulans (De Bary) Araaud is commonly known as "black yeast" and it has been given many names over the last 90 years because of the variety of environments in which it can develop. The following Table 1 shows the scientific classification of this yeast:
[0035] Table 1
Figure imgf000007_0001
[0036] Aureobasidium sp. is comprised in the epiphyte microbial flora of fruits and leaves of typical plants of the temperate regions. In the last thirty years, the use of Aureobasidium sp. as a biological control agent against fungal pathogens of fruits, in particular in the post-harvest phase, gave rise to a certain interest. In fact, among the numerous microorganisms that are potentially effective in reducing fruits rots, yeast or yeast-like fungi are particularly interesting for various reasons. First of all, the biological activity of these microorganisms does not derive from the production of antibiotic substances, as on the other hand occurs with many bacteria. Secondarily, these microorganisms do not have negative impacts from an environmental and toxicological point of view. Lastly, these microorganisms are easily cultivatable on a large scale, through the use of inexpensive and easily available substrates.
[0037] The strain according to the invention - namely the LI strain of A. pullulans - was isolated from the surface of "Redhaven" cultivar peaches grown in a peach orchard of the University of Bologna located in Cadriano (Man M., Martini C, Guidarelli M., Neri F., 2012. Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Biol Control 60: 132- 140). The strain was selected among different microorganisms in a study on identifying microbial antagonists. The LI strain was deposited on date 22 March 2017 at DBVPG (Industrial Yeasts Collection, Borgo XX Giugno, 74, 06121 Perugia, Italy) with number 39P. The LI strain is grown in laboratory on an NYDA agar substrate and the optimum growth temperature is 25°C. The NYDA agarized substrate is produced by dissolving the following ingredients in 1000 ml of distilled water: 8 grams of Nutrient Broth, 5 grams of Yeast Extract, 10 grams of dextrose and 15 grams of agar.
[0038] Already from the first selection steps, in vivo experiments showed that the LI strain has a strong attitude in resisting the development of some fruit phytopathogenic fungi, exhibiting a reduction of the disease greater than 50%. After performing preliminary tests, the LI strain was identified morphologically by observation under the microscope and molecular assays, such as the sequencing of the non-coding region of DNA (ITS = Internal transcribed spacer).
[0039] The outcomes of the observations under the microscope were comparable with the recognition keys (Barnett J.A., Payne R.W., Yarrow D., 2000. Yeasts: characteristics and identification. Cambridge University Press, UK pp 1139) of the species A. pullulans. The sequencing results confirmed the morphological identification, in fact the LI strain is 100% homologous to a strain of A. pullulans deposited at Gene Bank (CBS 584.75T, accession no. FJ150906).
[0040] Figure 1 shows yeast-like cells of the LI strain of A. pullulans, photographed under the Scanning Electron Microscope (SEM), which cells were grown on a peach of the aforesaid "Redhaven" cultivar.
[0041] Figure 2 shows the 100% homology between the ITS sequence of the LI strain of A. pullulans and a corresponding ITS sequence of the CBS 584.75T strain of A. pullulans. The ITS sequence of the LI strain of A. pullulans was further disclosed in a sequence listing (drawn up in compliance with WIPO Standard ST.25), which is attached to the present patent application and in which the aforesaid ITS sequence is named as SEQ ID NO: 1.
[0042] The studies on the LI strain continued, inasmuch as the possibility of applying microbial antagonists (such as the LI strain) to fruits before and after harvesting, in order to contain the development of fungal diseases, would enable known treatment methods to be partially replaced that are based on the use of synthetic fungicides. In this manner, it would be possible to reduce both the environmental impact and hygiene and sanitary problems due to the presence of plant protection products on fruits intended for fresh consumption. The LI strain has shown a certain effectiveness in reducing the occurrence of rots on fruits after harvesting, namely during the phase of conservation and marketing. In particular, the application of the LI strain on the fruits has reduced vegetable pathologies such as grey mould, brown rot and green-blue mould on apples, actinidia and oranges.
[0043] In order to improve the effectiveness of the LI strain, also in view of a possible formulation for possible marketing, the action mechanisms of the LI strain were examined more closely (Di Francesco, A., Ugolini, L, Lazzeri, L, Mari, M., 2015a. Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism action against postharvest fruit pathogens. Biol. Control 81: 8-14; Di Francesco, A., Roberti, R., Martini, M., Baraldi, E., Mari, M., 2015b. Activities of Aureobasidium pullulans cell filtrates against Monilinia laxa of peaches. Microbiol Research 181: 61-67).
[0044] In this step of in-depth study, the Inventors discovered the ability to metabolize the asparagine exhibited by the LI strain. In particular, it was observed that the LI strain used asparagine as a nutrient, producing a resulting accumulation of aspartic acid in the substrate (5% peach juice inoculated with the LI strain).
[0045] In the light of the obtained results, the Inventors decided to extend the experimental investigation to the potatoes. The asparagine is in fact one of the main amino acids contained in the potatoes and is (as highlighted previously) an important precursor of the acrylamide. As the problem is known of the formation of acrylamide in potatoes following high temperature thermal treatment (frying), the Inventors evaluated the possible ability of the LI strain to reduce the asparagine content in the potatoes in order to reduce the formation of acrylamide in transformed potatoes, such as fried potatoes of "French fries" type (stick-shaped) and fried potatoes of "chips" type (thin and crisp slices). In the following Example 1 a preliminary experimental test is disclosed that was conducted on a potato-based preparation.
[0046] Example 1 - Reduction of the asparagine content in a potato-based preparation by contact with LI strain of Aureobasidium pullulans
A homogenized potato product was prepared, by using a homogenizer of known type. A suspension in distilled water of the LI strain of Aureobasidium pullulans was prepared having a concentration (of yeast cells) of 108 CFU/ml. The aforesaid suspension is an example of a liquid formulation of the composition according to the invention. The suspension can be prepared by using tap water instead of distilled water. This liquid formulation is conservable stably (i.e. maintainable alive) for a maximum time of 120 hours at a temperature of 4 °C. A part of the homogenized potato product was placed in the LI strain suspension, by using an amount of homogenized potato product and of suspension such as to obtain a 1 : 1 (w/v) ratio. This sample was called "LI". A remaining part of the homogenized potato product (quantitatively equal to the LI sample) was not placed in contact with the LI strain suspension, but with an equal volume of water. This control sample was called "Test". The homogenized potato product was left in contact with the LI strain suspension for a time (contact time) of 30 minutes and at a temperature of 25°C. In parallel, The Test sample (homogenized potato product, without LI strain) was left for 30 minutes at a temperature of 25°C. After the aforesaid contact time elapsed, the extraction and analysis of free amino acids were made. The samples were diluted with water in a 1 :5 (w/v) ratio, subjected to ultrasound for 20 minutes and the liquid phase extract was recovered by centrifugation and was subsequently filtered (0.22 μιτι) to eliminate the yeast.
Aliquots of the water extracts of the LI sample and of the Test sample were analyzed by HPLC-DAD subject to derivatization, using apparatuses and methods as shown in Di Francesco, A., Ugolini, L, D Aquino, S., Pagnotta E., Mari, M., 2017. Biocontrol of Monilinia laxa by Aureobasidium pullulans strains: insights on competition for nutrients and space. Internat. J Food Microbiol 248: 32-38, in order to determine the quali- quantitative composition in amino acids in the LI sample and in the Test sample. The chromatograms obtained by the HPLC analysis are shown in Figure 3 and in Figure 4, with regard to the Test sample and the LI sample. The peaks in the chromatograms are indicated as: 1, aspartic acid; 2, glutamic acid; 3, asparagine; 4, serine; 5, glutamine; 6, threonine; 7, alanine; 8, arginine; 9 valine; 10, isoleucine/ phenylalanine; 11, leucine/lysine; SI, internal standard. The analysis of the Test sample (Figure 3) highlighted the peak of the asparagine (peak 3, in grey), which is on the other hand absent in the - chromatogram obtained from the analysis of the potato sample that was in contact with the LI strain. Similarly to what was observed experimentally in the case of peach juice (used as growth substrate for the LI strain), also in this case the metabolism of the asparagine by the yeast causes a consequent increase in aspartic acid, as it can be highlighted by the area of the peak relating to the aspartic acid (peak 1), which results to be significantly greater in the LI sample (Figure 4) than in the Test sample (Figure 3). The areas of the peaks relating to the other amino acids on the other hand remain nearly unchanged.
[0047] The results obtained in the Example 1 confirmed the ability of the LI strain of Aureobasidium pullulans to reduce the amount of asparagine contained in a substrate that is different from a vegetable juice (peach juice), such as a potato-based preparation (homogenized potato and water product).
[0048] In particular, it was seen that, in a contact time of 30 minutes, the strain according to the invention significantly metabolized the asparagine (which decreased from 652.0 mg/kg in the Test sample to less than 4.5 mg/kg in the LI sample, a value at the limit of detectability of the quantitative analysis by HPLC), causing a consequent increase in the aspartic acid (which increased from 395.4 mg/kg in the Test sample to 988.7 mg/kg in the LI sample).
[0049] The test conducted on the homogenized potato product was repeated on potatoes cut into pieces, as disclosed in the following Example 2. In particular, the potatoes were cut into thin slices, so as to simulate the fried potatoes of "chips" type.
[0050] Example 2 - Reduction of the acrylamide content in fried potatoes of "chips" type by treatment with the LI strain of Aureobasidium pullulans before the frying step. The potatoes were peeled, cut so as to obtain thin slices measuring about 1 mm and divided into two batches. One batch was immersed for 30 minutes (contact time) in a suspension of the LI strain of Aureobasidium pullulans in distilled water in a 3 x 106 CFU/ml concentration (sample treated with LI strain), in a 1 :2 w/v ratio. The aforesaid suspension constitutes an example of a liquid formulation of the composition according to the invention. The suspension can be prepared by using tap water instead of distilled water. The other batch was immersed for 30 minutes in sterile distilled water (untreated sample), maintaining the same w/v ratio (namely 1 :2 w/v ratio). After the contact time elapsed, the slices of both samples were washed under running water for 30 seconds, dried with absorbent paper and fried in a fryer (of known type) at 180°C for 150 seconds in high oleic sunflower oil. After the frying step, the slices of each batch were stored in a closed bag made of plastics until the moment of analysis of the acrylamide. The latter was conducted in a private laboratory by using known apparatuses and methods, namely gas chromatography-mass spectrometry (GC-MS). The results of the analysis conducted are shown in the following Table 2:
[0051] Table 2
Figure imgf000012_0001
[0052] From Table 2, it can be noted that the treatment with the strain according to the invention enabled the acrylamide content to be reduced by 83.45%.
[0053] Figure 5 is a photograph of the untreated sample after frying, whilst Figure 6 is a photograph of the sample treated with LI strain after frying. Comparing Figure 5 with Figure 6 it can be clearly noted that, in the untreated sample, during the frying step the Maillard reaction took place more intensely that in the sample treated with the LI strain, as the slices of Figure 5 appear darker (i.e. more browned) than the slices of Figure 6. This organoleptic element is considerable as a substantial confirmation of the analytical results of Table 2.
[0054] The experimental tests conducted by the Inventors clearly indicate that the LI strain of Aureobasidium pullulans, a composition incorporating the aforesaid strain and a method, according to which raw potatoes are treated with the aforesaid composition before being thermally treated, enable actually and effectively the formation of acrylamide in the fried potatoes to be reduced. This result is obtained without influencing the organoleptic features of the fried potatoes and in a substantially inexpensive manner. Therefore, it can be stated that the strain, the composition and the method according to the invention enable the previously illustrated drawbacks of the prior art to be overcome.
[0055] Variations on and/or additions to what has been disclosed above are moreover possible.
[0056] For example, although in Example 2 the composition according to the invention is disclosed as a liquid suspension of LI strain of A. pullulans, to a person skilled in the art the possibility appears clear of applying known methods (like those used, for example, in oenology and in the bakery products industry) to dehydrate the aforesaid suspension, so as to obtain a composition containing up to 95 - 97% of solid substance. In the aforesaid solid formulation, the strain according to the invention is vital, but remains metabolically inert until rehydration of the composition. The solid formulation of the composition according to the invention can be conserved and maintained stable for about 12-14 months.
[0057] Moreover, although in Example 2 the potatoes have been so cut as to obtain thin slices ("chips" or "crisps"), it is possible to treat with the strain, the composition and the method according to the invention also differently shaped potatoes, for example potatoes cut into the shape of a stick ("French fries" or "fries"). It should be further noted that, although in Example 2 a suspension of LI strain of Aureobasidium pullulans in distilled water was used in a 3 x 106 CFU/ml concentration, it is reasonable to state that the LI strain is also usable in a lower concentration, for example a 1 x 106 CFU/ml concentration (i.e. 106 CFU/ml).
[0058] Lastly, it should be noted that the ability of the LI strain of Aureobasidium pullulans to metabolize the asparagine was detected experimentally for the first time in a growth substrate different from potatoes. It can thus be hypothesized that the strain, the composition and the method according to the invention can be effectively used to reduce the amount of asparagine contained in foods and food matrices that are different from potatoes. In this manner, the strain, the composition and the method according to the invention could be used to reduce the amount of acrylamide that forms in foods and food matrices that are different from potatoes due to a thermal treatment at high temperatures, for example above 140°C.

Claims

1. Method for reducing the amount of asparagine that is contained in a raw food, so as to reduce the amount of acrylamide that forms in said food when said food is thermally treated at temperatures that are suitable for frying or cooking, said method comprising treating said raw food with the LI strain of Aureobasidium pullulans that is able to metabolize asparagine and deposited with number 39P.
2. Method according to claim 1, wherein the genome of said LI strain of Aureobasidium pullulans comprises the nucleotide sequence of SEQ ID NO: 1.
3. Method according to claim 1, or 2, wherein said treating comprises putting said raw food in contact with a composition containing said LI strain of Aureobasidium pullulans.
4. Method according to claim 3, wherein said composition contains said LI strain of Aureobasidium pullulans in a concentration of at least 106 CFU/ml.
5. Method according to claim 3, or 4, wherein said composition is a water suspension of said LI strain of Aureobasidium pullulans.
6. Method according to any one of claims 3 to 5, wherein, in said composition, said LI strain of Aureobasidium pullulans is dehydrated and can be rehydrated before use or at the time of use.
7. Method according to any one of claims 1 to 6, wherein said raw food is a food of vegetable origin.
8. Method according to claim 7, wherein said food of vegetable origin is a potato.
9. Method according to claim 8, as appended to any one of claims 3 to 6, wherein said putting in contact comprises immersing said potato in said composition.
10. Method according to claim 9, wherein said treating comprises maintaining said potato in contact with said composition for a period of at least 30 minutes.
11. Method according to claim 9, or 10, comprising removing said composition from said potato, after said treating.
12. Method according to claim 11, wherein said removing comprises washing said potato with running water.
13. Method according to claim 12, wherein said washing is performed over a period of at least 30 seconds.
14. Method according to any one of claims 11 to 13, comprising, after said removing, treating said potato thermally at temperatures that are suitable for frying.
15. Method according to claim 14, wherein said treating thermally is carried out at a temperature that is comprised between 160°C and 190°C.
16. Method according to claim 15, wherein said treating thermally is carried out at a temperature of 180°C.
17. Method according to any one of claims 14 to 16, wherein, through said treating thermally, a fried potato is obtained that has a reduced acrylamide content.
18. Method according to any one of claims 8 to 17, comprising peeling said potato and cutting said potato into pieces before said treating.
19. Use of the LI strain of Aureobasidium pullulans deposited with number 39P to reduce the amount of asparagine that is contained in a raw food, so as to reduce the amount of acrylamide that forms in said food when said food is thermally treated at temperatures that are suitable for frying or cooking, said LI strain of Aureobasidium pullulans being able to metabolize the asparagine.
20. Use according to claim 19, wherein the genome of said LI strain of Aureobasidium pullulans comprises the nucleotide sequence of SEQ ID NO: 1.
21. Use according to claim 19, or 20, wherein said LI strain of Aureobasidium pullulans is provided in a concentration of at least 106 CFU/ml.
22. Use according to any one of claims 19 to 21, wherein said LI strain oi Aureobasidium pullulans is in the form of water suspension.
23. Use according to any one of claims 19 to 22, wherein said LI strain of Aureobasidium pullulans is dehydrated and can be rehydrated before use or at the time of use.
24. Use according to any one of claims 19 to 23, wherein said raw food is a food of vegetable origin.
25. Use according to claim 24, wherein said food of vegetable origin is a potato.
26. Composition for reducing the amount of asparagine that is contained in a raw food, so as to reduce the amount of acrylamide that forms in said food when said food is thermally treated at temperatures that are suitable for frying or cooking, said composition comprising the LI strain of Aureobasidium pullulans that is able to metabolize the asparagine and deposited with number 39P.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2001078524A2 (en) * 2000-04-14 2001-10-25 Novo Nordisk Biotech, Inc. Enzymatic treatment of potato products
US20040086597A1 (en) * 2002-11-06 2004-05-06 Michael George Reduction of acrylamide formation in cooked starchy foods
WO2017004715A1 (en) * 2015-07-07 2017-01-12 Renaissance Bioscience Corp. Development of an asparagine-reducing yeast by adaptive evolution and uses thereof to reduce acrylamide formation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001078524A2 (en) * 2000-04-14 2001-10-25 Novo Nordisk Biotech, Inc. Enzymatic treatment of potato products
US20040086597A1 (en) * 2002-11-06 2004-05-06 Michael George Reduction of acrylamide formation in cooked starchy foods
WO2017004715A1 (en) * 2015-07-07 2017-01-12 Renaissance Bioscience Corp. Development of an asparagine-reducing yeast by adaptive evolution and uses thereof to reduce acrylamide formation

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Title
MARTA MARI ET AL: "Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Auriobasidium pullulans strains", BIOLOGICAL CONTROL, SAN DIEGO, CA, US, vol. 60, no. 2, 22 October 2011 (2011-10-22), pages 132 - 140, XP028439208, ISSN: 1049-9644, [retrieved on 20111029], DOI: 10.1016/J.BIOCONTROL.2011.10.013 *

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